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Third Edition
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February 2012
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Contents
Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Acknowledgment To Molecular Devices Consultants
And Customers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Editorial . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
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Chapter 1: Bioelectricity . . . . . . . . . . . . .
Electrical Potentials . . . . . . . . . . . . . . . .
Electrical Currents . . . . . . . . . . . . . . . . .
Resistors And Conductors . . . . . . . . . . . .
Ohms Law . . . . . . . . . . . . . . . . . . . . . .
The Voltage Divider . . . . . . . . . . . . . . . .
Perfect and Real Electrical Instruments. . .
Ions in Solutions and Electrodes . . . . . . .
Capacitors and Their Electrical Fields . . . .
Currents Through Capacitors . . . . . . . . . .
Current Clamp and Voltage Clamp . . . . . .
Glass Microelectrodes and Tight Seals . . .
Further Reading. . . . . . . . . . . . . . . . . . .
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Chapter 7: Transducers . . . . . . . . . . . . . . . . . . . . . . . .
Temperature Transducers for Physiological
Temperature Measurement . . . . . . . . . . . . . . . . . . . . .
Temperature Transducers for Extended
Temperature Ranges . . . . . . . . . . . . . . . . . . . . . . . . .
Electrode Resistance and Cabling Affect Noise
and Bandwidth . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
High Electrode Impedance Can Produce
Signal Attenuation . . . . . . . . . . . . . . . . . . . . . . . . . . .
Unmatched Electrode Impedances Increase
Background Noise and Crosstalk . . . . . . . . . . . . . . . . .
High Electrode Impedance Contributes to the
Thermal Noise of the System . . . . . . . . . . . . . . . . . . .
Cable Capacitance Filters out the High-Frequency
Component of the Signal . . . . . . . . . . . . . . . . . . . . . .
EMG, EEG, EKG, and Neural Recording . . . . . . . . . . . . .
Metal Microelectrodes . . . . . . . . . . . . . . . . . . . . . . . . .
Bridge Design for Pressure and Force Measurements . . .
Pressure Measurements . . . . . . . . . . . . . . . . . . . . . . .
Force Measurements . . . . . . . . . . . . . . . . . . . . . . . . .
Acceleration Measurements. . . . . . . . . . . . . . . . . . . . .
Length Measurements . . . . . . . . . . . . . . . . . . . . . . . .
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Self-Heating Measurements .
Isolation Measurements . . .
Insulation Techniques . . . . .
Further Reading . . . . . . . . .
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Preface
Molecular Devices is pleased to present you with the third edition of the
Axon Guide, a laboratory guide to electrophysiology and biophysics.
The purpose of this guide is to serve as an information and data
resource for electrophysiologists. It covers a broad scope of topics
ranging from the biological basis of bioelectricity and a description of
the basic experimental setup to a discussion of mechanisms of noise
and data analysis.
The Axon Guide third edition is a tool benefiting both the novice and the
expert electrophysiologist. Newcomers to electrophysiology will gain an
appreciation of the intricacies of electrophysiological measurements
and the requirements for setting up a complete recording and analysis
system. For experienced electrophysiologists, we include in-depth
discussions of selected topics, such as advanced methods in
electrophysiology and noise.
This edition is the first major revision of the Axon Guide since the
original edition was published in 1993. While the fundamentals of
electrophysiology have not changed in that time, changes in
instrumentation and computer technology made a number of the
original chapters interesting historical documents rather than helpful
guides. This third edition is up-to-date with current developments in
technology and instrumentation.
This guide was the product of a collaborative effort of many researchers
in the field of electrophysiology and the staff of Molecular Devices. We
are deeply grateful to these individuals for sharing their knowledge and
devoting significant time and effort to this endeavor.
David Yamane
Director, Electrophysiology Marketing
Molecular Devices
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Preface
10
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Introduction
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11
Introduction
12
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Editorial
Editor
Rivka Sherman-Gold, Ph.D.
Editorial Committee
Michael J. Delay, Ph.D.
Alan S. Finkel, Ph.D.
Henry A. Lester, Ph.D.1
W. Geoffrey Powell, MBA
Rivka Sherman-Gold, Ph.D.
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Editorial
Acknowledgements
The valuable inputs and insightful comments of Dr. Bertil Hille, Dr. Joe
Immel, and Dr. Stephen Redman are much appreciated.
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Contributors
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Contributors
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Bioelectricity
Electrical Potentials
A cell derives its electrical properties mostly from the electrical
properties of its membrane. A membrane, in turn, acquires its
properties from its lipids and proteins, such as ion channels and
transporters. An electrical potential difference exists between the
interior and exterior of cells. A charged object (ion) gains or loses
energy as it moves between places of different electrical potential, just
as an object with mass moves up or down between points of
different gravitational potential. Electrical potential differences are
usually denoted as V or V and measured in volts. Potential is also
termed voltage. The potential difference across a cell relates the
potential of the cells interior to that of the external solution, which,
according to the commonly accepted convention, is zero.
The potential difference across a lipid cellular membrane
(transmembrane potential) is generated by the pump proteins that
harness chemical energy to move ions across the cell membrane. This
separation of charge creates the transmembrane potential. Because the
lipid membrane is a good insulator, the transmembrane potential is
maintained in the absence of open pores or channels that can conduct
ions.
Typical transmembrane potentials amount to less than 0.1 V, usually 30
to 90 mV in most animal cells, but can be as much as 200 mV in plant
cells. Because the salt-rich solutions of the cytoplasm and extracellular
milieu are fairly good conductors, there are usually very small
differences at steady state (rarely more than a few millivolts) between
any two points within a cells cytoplasm or within the extracellular
solution. Electrophysiological equipment enables researchers to
measure potential (voltage) differences in biological systems.
Electrical Currents
Electrophysiological equipment can also measure current, which is the
flow of electrical charge passing a point per unit of time. Current (I) is
measured in amperes (A). Usually, currents measured by
electrophysiological equipment range from picoamperes to
microamperes. For instance, typically, 104 Na+ ions cross the
membrane each millisecond that a single Na+ channel is open. This
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Bioelectricity
current equals 1.6 pA (1.6 x 10-19 C/ion x 104 ions/ms x 103 ms/s; recall
that 1 A is equal to 1 coulomb (C)/s).
Two handy rules about currents often help to understand
electrophysiological phenomena: 1) current is conserved at a branch
point (Figure 1-1); and 2) current always flows in a complete circuit
(Figure 1-2). In electrophysiological measurements, currents can flow
through capacitors, resistors, ion channels, amplifiers, electrodes and
other entities, but they always flow in complete circuits.
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Bioelectricity
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Bioelectricity
potential for that ion. Since a typical cell membrane at rest has a much
higher permeability to potassium (PK) than to sodium, calcium or
chloride (PNa, PCa and PCl, respectively), the resting membrane potential
is very close to EK, the potassium reversal potential.
Ohms Law
For electrophysiology, perhaps the most important law of electricity is
Ohms law. The potential difference between two points linked by a
current path with a conductance G and a current I (Figure 1-5) is:
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23
Bioelectricity
When two resistors are connected in series, the same current passes
through each of them. Therefore the circuit is described by:
where E is the value of the battery, which equals the total potential
difference across both resistors. As a result, the potential difference is
divided in proportion to the two resistance values.
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Bioelectricity
Before making the measurement, the cell has a resting potential of Erp,
which is to be measured with an intracellular electrode of resistance Re.
To understand the effect of the measuring circuit on the measured
parameter, we will pretend that our instrument is a perfect voltmeter
(for example, with an infinite resistance) in parallel with a finite
resistance Rin, which represents the resistance of a real voltmeter or
the measuring circuit. The combination Re and Rin forms a voltage
divider, so that only a fraction of Erp appears at the input of the
perfect voltmeter; this fraction equals ErpRin/(Rin + Re). The larger Rin,
the closer V is to Erp. Clearly the problem gets more serious as the
electrode resistance Re increases, but the best solution is to make Rin as
large as possible.
On the other hand, the best way to measure current is to open the path
and insert an ammeter. If the ammeter has zero resistance, it will not
perturb the circuit since there is no IR-drop across it.
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1.
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Bioelectricity
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Bioelectricity
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Bioelectricity
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This is just Ohms law, of course; but when the membrane capacitance
is in the circuit, the voltage is not reached immediately. Instead, it is
approached with the time constant , given by:
Thus, the charging time constant increases when either the membrane
capacitance or the resistance increases. Consequently, large cells, such
as Xenopus oocytes that are frequently used for expression of genes
encoding ion-channel proteins, and cells with extensive membrane
invigorations, such as the T-system in skeletal muscle, have a long
charging phase.
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Bioelectricity
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Bioelectricity
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Further Reading
Brown, K. T., Flaming, D. G. Advanced Micropipette Techniques for Cell
Physiology. John Wiley & Sons, New York, NY, 1986.
Hille, B., Ionic Channels in Excitable Membranes. Second edition.
Sinauer Associates, Sunderland, MA, 1991.
Horowitz, P., Hill, W. The Art of Electronics. Second edition. Cambridge
University Press, Cambridge, UK, 1989.
Miller, C., Ion Channel Reconstitution. C. Miller, Ed., Plenum Press, New
York, NY, 1986.
Sakmann, B., Neher, E. Eds., Single-Channel Recording. Plenum Press,
New York, NY, 1983.
Smith, T. G., Lecar, H., Redman, S. J., Gage, P. W., Eds. Voltage and
Patch Clamping with Microelectrodes. American Physiological Society,
Bethesda, MD, 1985.
Standen, N. B., Gray, P. T. A., Whitaker, M. J., Eds. Microelectrode
Techniques: The Plymouth Workshop Handbook. The Company of
Biologists Limited, Cambridge, England, 1987.
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Bioelectricity
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monitor is quieter than a color monitor). Note that shielding may bring
its own penalties, such as introducing other kinds of noise or degrading
ones bandwidth (see Chapter 11: Noise in Electrophysiological
Measurements on page 259). Do not assume that commercial
specifications are accurate. For example, a DC power supply for the
microscope lamp might have considerable AC ripple, and the shielded
lead connecting the microelectrode preamplifier to the main amplifier
might need additional shielding. Solution-filled perfusion tubing
entering the bath may act as an antenna and pick up radiated noise. If
this happens, shielding of the tubing may be required. Alternatively, a
drip-feed reservoir, such as is used in intravenous perfusion sets, may
be inserted in series with the tubing to break the electrical continuity of
the perfusion fluid. Never directly ground the solution other than at the
ground wire in the chamber, which is the reference ground for the
amplifier and which may not be the same as the signal ground used for
shielding purposes. Further suggestions are given in Line-frequency
Pick-up (Hum) on page 200.
Magnetically-induced Pickup
Magnetically-induced pickup arises whenever a changing magnetic flux
passes through a loop of wire, thereby inducing a current in the wire. It
most often originates in the vicinity of electromagnets in power
supplies, and is usually identified by its non-sinusoidal shape with a
frequency that is a higher harmonic of the line frequency. This type of
interference is easily reduced by moving power supplies away from
sensitive circuitry. If this is not possible, try twisting the signal wires
together to reduce the area of the loop cut by the flux, or try shielding
the magnetic source with mu-metal.
Ground-loop Noise
Ground-loop noise arises when shielding is grounded at more than one
place. Magnetic fields may induce currents in this loop. Moreover, if the
different grounds are at slightly different potentials, a current may flow
through the shielding and introduce noise. In principle, ground loops
are easy to eliminate: simply connect all the shields and then ground
them at one place only. For instance, ground all the connected shields
at the signal ground of the microelectrode amplifier. This signal ground
is, in turn, connected at only one place to the power ground that is
provided by the wall socket. In practice, however, one is usually
frustrated by ones ignorance of the grounding circuitry inside electronic
apparatuses. For example, the shielding on a BNC cable will generally
be connected to the signal ground of each piece of equipment to which
it is attached. Furthermore, each signal ground may be connected to a
separate power ground (but not on Axon Cellular Neuroscience
amplifiers). The loop might be broken by lifting off the BNC shielding
and/or disconnecting some power grounds (although this creates
hazards of electrocution!). One could also try different power sockets,
because the mains earth line may have a lower resistance to some
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sockets than others. The grounds of computers are notorious for noise.
Thus, a large reduction in ground-loop noise might be accomplished by
using optical isolation (see Chapter 9: Acquisition Hardware on
page 231) or by providing the computer with a special power line with
its own ground.
The logical approach to reducing noise in the setup is to start with all
equipment switched off and disconnected; only an oscilloscope should
be connected to the microelectrode amplifier. First, measure the noise
when the headstage is wrapped in grounded metal foil. Microelectrode
headstages should be grounded through a low resistance (for instance,
1 M), whereas patch-clamp headstages should be left open circuit.
This provides a reference value for the minimum attainable radiative
noise. Next, connect additional pieces of electronic apparatuses while
watching for the appearance of ground loops. Last, install an electrode
and add shielding to minimize radiative pickup. Finally, it should be
admitted that one always begins noise reduction in a mood of optimistic
rationalism, but invariably descends into frustrating empiricism.
Equipment Placement
While the placement of equipment is directed by personal preferences,
a brief tour of electrophysiologists common preferences may be
instructive. Electrophysiologists tend to prefer working alone in the
corners of small rooms. This is partly because their work often involves
bursts of intense, intricate activity when distracting social interactions
are inadmissible. Furthermore, small rooms are often physically quieter
since vibrations and air currents are reduced. Having decided upon a
room, it is usually sensible to first set up the microscope and its
intimate attachments, such as the chamber, the manipulators and the
temperature control system (if installed). The rationale here is that
ones first priority is to keep the cells happy in their quiescent state,
and ones second priority is to ensure that the act of recording from
them is not consistently fatal. The former is assisted by a good
environment, the latter by good optics and mechanics. Working
outward from the microscope, it is clearly prudent to keep such things
as perfusion stopcocks and micromanipulator controllers off the
vibration isolation table. Ideally, these should be placed on small
shelves that extend over the table where they can be accessed without
causing damaging vibrations and are conveniently at hand while
looking through the microscope.
Choice and placement of electronics is again a matter of personal
preference. There are minimalists who make do with just an amplifier
and a computer, and who look forward to the day when even those two
will coalesce. Others insist on a loaded instrument rack. An oscilloscope
is important, because the computer is often insufficiently flexible.
Furthermore, an oscilloscope often reveals unexpected subtleties in the
signal that were not apparent on the computer screen because the
sample interval happened not to have been set exactly right. The
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List of Equipment
Table 2-1 Traditional Patch-Clamp Setup
Item
Suggested Manufacturers
Newport
Micro-g (Technical Manufacturing Corp.)
Microscope, inverted
Carl Zeiss
Leica Microsystems
Nikon
Olympus
Micromanipulators
hydraulic
motorized
piezoelectric
44
Patch-clamp amplifiers
Molecular Devices
(Axon Cellular Neuroscience)
Oscilloscopes
Tektronix
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coaters
hydrophobic coating
Microelectrode holders
Suggested Manufacturers
Garner Glass
Friedrich & Dimmock
Sutter Instrument
Sutter Instrument
Narishige International USA
homemade - based on:
Carl Zeiss metallurgical microscope
Olympus CH microscope
Narishige International USA
Dow Corning Sylgard 184
Q-dope
Molecular Devices
(Axon Cellular Neuroscience)
E. W. Wright
Computers
Suggested Manufacturers
Carl Zeiss
Vibratome
Vibratome
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Item
Suggested Manufacturers
Newport
Micro-g (Technical Manufacturing Corp.)
Microscope
Carl Zeiss
Leica Microsystems
Nikon
Olympus
45
Suggested Manufacturers
Micromanipulators
mechanical
hydraulic
piezoelectric
Microelectrode amplifiers
Molecular Devices
(Axon Cellular Neuroscience)
Oscilloscopes
Tektronix
Electrode fabrication
glass
pullers
Garner Glass
Friedrich & Dimmock
Sutter Instrument
David Kopf
Sutter Instrument
GlasswoRx
Microelectrode holders
Molecular Devices
(Axon Cellular Neuroscience)
E. W. Wright
Computers
Suggested Manufacturers
Photomultipliers
Hamamatsu
Imaging systems
Molecular Devices
Further Reading
Conventional intra- and extracellular recording from brain
slices
Dingledine, R. Ed. Brain Slices. Plenum Press, New York, NY, 1983.
Geddes, L. A. Electrodes and the Measurement of Bioelectric Events.
Wiley Interscience, 1972.
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Patch-slice recording
Edwards, F. A., Konnerth, A., Sakmann, B., Takahashi, T. A thin slice
preparation for patch clamp recordings from neurons of the mammalian
central nervous system. Pflugers Arch. 414: 600612, 1989.
Blanton, M. G., Lo Turco, J. J., Kriegstein, A. Whole cell recording from
neurons in slices of reptilian and mammalian cerebral cortex. J.
Neurosci. Meth. 30: 203210, 1989.
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Extracellular Recording
The most straight-forward electrophysiological recording situation is
extracellular recording. In this mode, field potentials outside cells are
amplified by an AC-coupled amplifier to levels that are suitable for
recording on a chart recorder or computer. The extracellular signals are
very small, arising from the flow of ionic current through extracellular
fluid (see Chapter 1: Bioelectricity on page 17). Since this saline fluid
has low resistivity, and the currents are small, the signals recorded in
the vicinity of the recording electrode are themselves very small,
typically on the order of 10500 V.
The most important design criterion for extracellular amplifiers is low
instrumentation noise. Noise of less than 10 V peak-to-peak (Vp-p) is
desirable in the 10 kHz bandwidth. At the same time, the input bias
current3 of the amplifier should be low (< 1 nA) so that electrodes do
1.
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49
not polarize. Ideally, the amplifier will have built-in high-pass and
low-pass filters so that the experimenter can focus on the useful signal
bandwidth.
Single-Cell Recording
In single-cell extracellular recording, a fine-tipped microelectrode is
advanced into the preparation until a dominant extracellular signal is
detected. This will generally be due to the activity of one cell. The
microelectrode may be made of metal, for example, glass-insulated
platinum, or it may be a saline-filled glass micropipette.
While not specifically targeted at extracellular recording, the
Axoclamp 900A and the MultiClamp 700B amplifiers are particularly
suitable for single-cell extracellular recording if the extracellular
electrode is a microelectrode of several megohms or more. The input
leakage current of these amplifiers is very low and headstages are
designed to directly accommodate the micropipette holder. If
necessary, capacitance compensation can be used to speed up the
response. The x100 AC-coupled output signal of the MultiClamp 700B
amplifier, in particular, is useful for measuring small extracellular
signals, because up to 2000x further amplification can be applied to the
signal with the built-in output gain. Thus the total amplification is
200,000x, allowing for easier measurement of extracellular potentials
of less than 1 mV.
The Axon CyberAmp 380 programmable signal conditioner amplifier
has special ultra low-noise probes suitable for extracellular recording.
These probes do not have the special features of the Axoclamp 900A
and the MultiClamp 700B amplifiers, but for low-resistance electrodes,
from tens of ohms up to a few hundred kilohms, these ultra low-noise
probes have superior noise characteristics. The AI 402 x50 probe for
the CyberAmp 380 amplifier contributes less noise than the thermal
noise of a 250 resistor. Electrodes can be connected directly to the
CyberAmp 380 amplifier without using a separate low-noise probe. In
this case, the additional noise due to the amplifier will still be very
lowless than the thermal noise of a 5 k resistor. If electrodes are
directly connected to the main instrument, there is always a risk of
picking up line-frequency noise or noise from other equipment. Using a
probe located very close to the electrode greatly reduces the probability
that it will pick up extraneous noise.
3.
50
In the ideal operational amplifier (op amp), no current flows into the inputs.
Similarly, in the ideal transistor, no current flows into the gate. In practice,
however, amplifying devices always have an input current. This current is
commonly known as the input bias current.
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Multiple-cell Recording
In multiple-cell extracellular recording, the goal is to record from many
neurons simultaneously to study their concerted activity. Several
microelectrodes are inserted into one region of the preparation. Each
electrode in the array must have its own amplifier and filters. If tens or
hundreds of microelectrodes are used, special fabrication techniques
are required to produce integrated pre-amplifiers. If recording is
required from up to 16 sites, two CyberAmp 380 amplifiers can be
alternately used with one 16-channel A/D system such as the Digidata
1440A digitizer.
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Bridge Balance
Can the intracellular potential be measured if the current injected down
the micropipette is a variable waveform? Without special compensation
circuitry, the answer is no. The variable current waveform causes a
corresponding voltage drop across the micropipette. It is too difficult to
distinguish the intracellular potential from the changing voltage drop
across the micropipette. However, special compensation circuitry can
be used to eliminate the micropipette voltage drop from the recording.
The essence of the technique is to generate a signal that is proportional
to the product of the micropipette current and the micropipette
resistance. This signal is then subtracted from the buffer amplifier
output (Figure 3-3). This subtraction technique is commonly known as
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53
eliminated (Figure 3-4). At this point, the circuit is balanced and the
micropipette resistance can be read from the calibrated readout of the
Bridge Balance control. The same technique can even be used after the
micropipette has penetrated the cell. Details of how to achieve this are
described in the Axoclamp 900A and MultiClamp 700B manuals.
Junction Potentials
A second extraneous contributor to the measured voltage at the output
of the micropipette buffer amplifier is the sum of the junction
potentials. Junction potentials occur wherever dissimilar conductors are
in contact. The largest junction potentials occur at the liquid-metal
junction formed where the wire from the amplifier input contacts the
electrolyte in the micropipette and at the liquid-liquid junction formed
at the tip of the micropipette. The sum of all of the junction potentials
can be eliminated by introducing a single DC potential of the opposite
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Track
A special form of automatic offset compensation is available in many
patch clamp amplifiers, including the Axopatch-1 and the Axopatch
200 amplifier series. In Axopatch amplifiers this technique is called
Track. In some instruments of other manufacturers it is called
Search. The Track circuit is used only during seal formation. During
seal formation the tip comes in and out of contact with the membrane
and rapid shifts in the tip potential of the headstage sometimes occur.
These potential shifts lead to equivalent rapid shifts in the current
record, which may exceed the dynamic range of the headstage. Since
the headstage itself saturates, AC coupling at the oscilloscope or any
other form of external DC offset removal will not be useful. Instead, the
Track circuit dynamically adjusts the offset potential of the micropipette
itself, so that the current is forced to be zero on average. It is important
that the Track circuit be switched off before recording of ionic currents
commences since the Track circuit introduces the same type of
distortion produced by AC coupling.
Current Monitor
Current-clamp circuits include a monitor output proportional to the
current waveform. In some current-clamp amplifiers, the monitor
output is merely a scaled version of the control voltage driving the
current-clamp circuit. As long as the micropipette resistance is
moderate and the current-clamp circuit output does not exceed its
linear operating range, this technique is accurate. However, if the
micropipette resistance is very high and the current-clamp circuitry
saturates, the actual current through the micropipette will be less than
the expected current. This simple monitor output will not indicate the
failure to pass the expected current. A better technique is to actually
measure the current by monitoring the voltage drop across a resistor in
series with the micropipette (Figure 3-5). This is a superior technique
used in all of Axons micropipette current-clamp amplifiers, including
the Axoclamp 900A and MultiClamp 700A and 700B.
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1. The input lead to the virtual ground is very sensitive and easily
picks up line-frequency and other interference.
2. It is extremely difficult to use two microelectrode amplifiers in
the same preparation bath if one or both of them uses a
virtual-ground current-measurement circuit. If one amplifier
uses a virtual ground, the individual micropipette currents will
be difficult to identify because the virtual-ground current
monitor measures the summed current from all sources. If two
amplifiers use a virtual ground, large DC-error currents will flow
between the virtual-ground circuits because of the small
differences in offset voltages between the two circuits. This is
equivalent to the problem that would exist if two voltage sources
were connected in parallel.
3. The virtual-ground probe is just one more box of electronics that
has to be mounted in the crowded space around the preparation
bath.
Nevertheless, the virtual ground technique is still used in some
circumstances, most commonly with high-voltage two-electrode
voltage-clamp amplifiers, because of the technical difficulty of making a
series current-measurement circuit operate at high voltages.
The Bath Error Potentials section below describes an important
variation on the virtual-ground design to eliminate the voltage error
due to current flow through the grounding electrode.
Capacitance Compensation
The high-frequency performance of the micropipette amplifier is
compromised by the presence of capacitance at the amplifier input.
This capacitance comes from several sources: the capacitance across
the glass wall (transmural capacitance) of the immersed part of the
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Leakage Current
Ideally, a micropipette headstage should pass zero current into the
micropipette when the current command is at zero. In practice, there is
a leakage current created from two sources. The first is the inherent
bias current of the operational amplifiers input. This is usually of the
order of a few picoamps or less. The second is the current that flows
through the current injection resistor (Ro in Figure 3-2) because of
voltage offsets in the current-control circuitry. A trim potentiometer can
be used to reduce this offset. In fact, an optimal setting can be found
where a small offset is left across Ro so that the resistor current
compensates the operational amplifier bias current and leaves a net
zero current through the micropipette. However, the leakage current
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sum of the transmembrane potential (Vm) and the bath potential (Vb).
Problems arise if the product of the clamp current (I2) and Rb is
significant. For example, for I2 = 5 A and Rb = 2 k, the error voltage
is 10 mV. In some experiments, a worst-case error of this magnitude
might be tolerable; but if the error were to be much greater, the
position of the peak of I-V curves and other responses would be
seriously affected.
To faithfully record Vm, either Vb must be made equal to or nearly equal
to zero, or the value of Vb must be independently measured and
subtracted from the potential recorded by ME1. There are several
methods to choose from:
1. The best method is to minimize Rb in the first place.
2. Electronically predict and subtract the error voltage. This
technique is known as series-resistance compensation.
3. Use an independent electrode to measure Vb and subtract this
value from V1.
4. Set up an independent clamping circuit to clamp Vb, measured
near the surface of the cell, to zero.
5. Set up an independent virtual-ground circuit to clamp Vb,
measured near the surface of the cell, to zero, while
simultaneously measuring the bath current.
Minimizing Rb
There are three main contributors to Rb:
1. The cell access resistance from the membrane surface to the
bath.
2. The resistance of an agar bridge, if used.
3. The resistance of the grounding wire.
Cell Access Resistance
The access resistance to a sphere is given by:
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where length refers to the length of the agar bridge and area is
the cross-sectional area of the agar bridge.
For a 1 cm long agar bridge with a 2 mm internal diameter (ID)
filled with Ringers solution:
4.
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Series-Resistance Compensation
This sophisticated technique is used to minimize the effects of the
electrode resistance in a whole-cell voltage clamp. It is described later
in this chapter and is of paramount importance when there is no
alternative available. However, series-resistance compensation is never
completely effective. The other methods discussed in this section are
more reliable and easier to apply when it comes to eliminating the
voltage error across Rb.
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There are several problems with this technique. First, if the output is
used to record the bath current, it is usual to ignore the Rb1 term
(because its value is unknown) and to assume that
Thus, there is a small error equal to Rb1/(Rb1 + Rf ). Second, if there are
fluid-filled tubes connected to the bath, and if some of these run
outside of the Faraday cage, they will act as antennas and conduct a lot
of hum current into the bath that will be recorded by the virtual-ground
circuit. Third, if it is desired to use more than one amplifier with the
same preparation, it is essential that no more than one of them uses a
virtual-ground circuit to clamp the bath potential. This is not a serious
problem, since it is easy to make one virtual-ground serve the needs of
both amplifiers. The problem is serious, however, if one of the
amplifiers introduces command signals via the virtual-ground bath
clamp. This is a practice employed by some manufacturers because in
some circumstances it is easier to design the electronics for this mode
of operation. However, if command potentials are introduced via the
bath electrode, it is extremely difficult to perform experiments using
two amplifiers, for example, whole-cell patch clamp of two fused cells.
When used with consideration for the measurement error, and if hum
pick up is avoided, a virtual-ground circuit to measure IBATH offers
excellent performance.
Summary
As a first priority, we recommend that the value of Rb be minimized by
the techniques described above. If it is still considered to be important
to compensate for the IR voltage drop, a virtual-ground headstage
(such as the VG-9A series) can be used to clamp the bath potential, or
a unity-gain voltage follower headstage (such as the HS-9A and
HS-2A series) can be used to record and subtract the bath potential.
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duration can also be controlled via its remote buzz accessory unit. The
duration used should be long enough for penetration, but short enough
to prevent damage to the cell. The mechanism of penetration is
unknown, but it may involve attraction between the charge at the tip of
the micropipette and bound charges on the inside of the membrane.
The second electronic alternative is to drive a large positive or negative
current step into the cell.
In the Axoclamp 900A amplifier, both the duration and height of the
current pulse are adjustable. Again, the mechanism of penetration is
not known. One cannot even predict whether a positive or a negative
pulse will be more effective.
None of the three methods described here can be described as best.
For a particular cell type, the researcher must experiment to find the
method that works best with those cells.
Command Generation
In general, current and voltage commands arise from a variety of
sources: internal oscillators in the instrument, internal DC current or
voltage controls, external input from a computer, etc. These commands
are usually summed in the instrument so that, for example, an
externally generated step command can be superimposed on the
internally generated holding potential.
An important consideration when using externally generated commands
is the attenuation provided by the instruments command input.
Manufacturers attenuate the external command so that the effects of
noise and offset in the external signal generator are minimized. Noise is
particularly common on the analog output of most computer interfaces.
To illustrate the problem, imagine a micropipette amplifier used for
current injection into cells that has a current passing capacity of
100 nA maximum. Compare the situation for two different values of
the command input attenuation. In the first case, the amplifier passes
1 nA for each millivolt from the computer. If there is a 2 mV of wideband digital noise on the output of the D/A converter, this will generate
2 nA of noise in the recording a substantial fraction of the usable range.
Further, if the D/A converter had a 3 mV offset, then instead of a
command of zero generating 0 nA, it would generate 3 nA. In the
second case, the amplifier passes 1 nA for each 100 mV from the
computer. This time, the 2 nA of noise from the D/A converter only
generates 20 pA of noise in the recording, and the 3 mV offset error
only generates a 30 pA current error. The full 100 nA range of the
amplifier can still be controlled, since most D/A converters produce up
to 10 V.
All of the Axon Conventional Electrophysiology current-clamp and
voltage-clamp instruments from Molecular Devices use the maximum
possible attenuation so that the 10.24 V output from the D/A
converter corresponds to the normal maximum operating limits of the
instrument.
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Unlike the ideal case, there is a finite time required to charge the cell
capacitance. The time constant for the current and potential transients
is = Rp2Cm/, where Rp2 is the resistance of ME2. If was infinite, or if
Rp2 was zero, the response would approach the ideal case.
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Error
The steady-state membrane potential (Vm) after a step change in the
command voltage (Vcmd) is:
Increasing the clamp gain decreases the time constant for the step
response. For example, if Rp2 = 10 M, Cm = 1000 pF and = 100, the
time constant is 100 s.
Stated differently, increasing the clamp gain also increases the
bandwidth with which Vm can follow changes in Vcmd. The -3 dB
frequency of the bandwidth is:
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Stability
The voltage clamp circuit shown in Figure 3-12 is unconditionally
stable. The membrane capacitance provides a 90 phase shift, which is
required for stability in all negative feedback circuits. Unfortunately,
other factors combine to make the circuit unstable at high clamp gains.
The coupling capacitance (Cx) between the micropipettes is extremely
destabilizing. Values as small as 0.01 pF can lead to oscillation if has
a magnitude of several hundred or more. There are several ways to
minimize this coupling capacitance. The two best ways are by:
1. Introducing the two micropipettes into the preparation at a wide
angle, preferably greater than 90.
2. Placing a grounded metal shield between the two micropipettes.
This shield should be large enough to block all line-of-sight
pathways between the two micropipettes and their holders.
Another destabilizing factor is the non-ideal nature of the membrane.
In Figure 3-11, the membrane is simply modeled as a parallel resistor
and capacitor. In practice, a distributed model applies. The capacitance
elements are themselves non-ideal; they should be modeled by an
ideal capacitor with a series-resistance component. For real
membranes, the phase shift at high frequencies is less than 90. In the
Axoclamp 900A amplifier, a phaseshift control is included to allow the
user to empirically introduce a phase lag to the circuit to build the total
high-frequency phase shift up to 90.
The input capacitance of the voltage-recording micropipette (ME1) adds
another frequency-dependent variable into the system that also tends
to decrease the stability. The effect of this input capacitance is usually
minimized by carefully adjusting the capacitance neutralization control
to maximize the bandwidth of ME1.
Small instabilities in the voltage clamp usually show up as an overshoot
in the current and voltage responses. Full instability shows up as a
continuous oscillation that can destroy the cell. In some voltage
clamps, special circuits are used to detect oscillations and take
appropriate action to avoid damage to the cell. The Axoclamp 2 series
and the GeneClamp 500 amplifiers do not include an oscillation guard
circuit. The Axoclamp 900A amplifier includes an oscillation detection
feature that automatically reduces the voltage clamp gain, protecting
the cells from harm.
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Noise
There are several sources of noise in a two-electrode voltage-clamp
circuit, but the most important is the thermal and excess noise of the
input micropipette, ME1. To the voltage-clamp circuit, this noise
appears as an extra command signal. It is imposed across the
membrane and leads to a noise current. Because of the membrane
capacitance, this noise current increases directly with frequency in the
bandwidths of interest. The magnitude of the noise is worst in cells with
large Cm values.
Since the current noise increases with frequency, a single-pole filter is
inadequate. An active two-pole filter is required at minimum; while a
four-pole filter is preferred. The Axoclamp 900A amplifier includes a
four-pole filter with a corner frequency up to 30 kHz on each output.
The user can choose between a Bessel and a Butterworth filter.
Micropipette Selection
Ideally, both micropipettes should have very small resistances. The
resistance of ME1 should be small to minimize the micropipette noise,
and the resistance of ME2 should be small to maximize the product K.
However, low-resistance micropipettes are more blunt than
high-resistance micropipettes and do more damage to the cell. In
general, it is more important to use a low resistance micropipette for
ME2. If its value is high, the amplifiers power supply will saturate at a
lower current, causing a voltage-clamp error because the ME2
headstage cannot inject enough current to hold the membrane voltage
clamped. As a result, the recorded data will be of dubious merit. A
relatively high-resistance micropipette for ME1 has its problems
increased noise and reduced bandwidthbut at least it does not
introduce an error.
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Thus for a given GT, if Cm is small, Ti must be small (for example, fs must
be large).
For example, if GT = 1 nA/mV and Cm = 100 pF, then Ti must be 100 s
for optimum response. If Ti is greater than 100 s, the step response
will overshoot and at 200 s the clamp will oscillate. (Note that in
Axoclamp amplifiers, Ti = 100 s corresponds to a sampling rate of
3 kHz because the duty cycle (discussed later) is 30%.)
If Ti in this example cannot be as short as 100 s because the
micropipette response is too slow, then a lower value of GT will have to
be used to maintain optimum response.
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compromise is a duty cycle equal to 0.3. This is the value used in the
Axoclamp amplifiers.
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After Vcmd steps to V1, a steady-state current, Im, flows in the circuit. The
membrane potential is equal to Vcmd - ImRa,eff. After the step change in
the command potential, Im and Vm settle exponentially to their steadystate values with =[Ra,effRm/(Ra,eff + Rm)]Cm, but since in general Rm
>> Ra,eff, a good approximation is Ra,effCm.
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Series-Resistance Compensation
In the ideal experiment, the resistance of the patch micropipette in
whole-cell experiments would be zero. In this case, the time resolution
for measuring membrane currents and changing the membrane voltage
would be limited only by the speed of the electronics (typically just a
few microseconds).
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Supercharging (prediction)
Another technique for speeding up the response to a command step is
the supercharging technique, also known as prediction. In contrast
to the correction method of series-resistance compensation,
prediction is an open-loop method. That is, there is no feedback and
there is little risk of oscillations.
Supercharging is accomplished by adding a brief charging pulse at the
start and the end of the command voltage pulse. This means that
initially the membrane is charging towards a larger final value than
expected (Figure 3-16). In its crudest form, the charging-pulse
amplitude or duration are adjusted empirically by the investigator so
that the membrane potential does not overshoot. Since the membrane
potential is not directly observable by the user, this is accomplished by
adjusting the controls until the current transient is as brief as possible.
Once the optimum setting for one step size has been found, the size of
the supercharging pulse for all other step sizes can be calculated by the
computer. Since the large, transient supercharging current has to be
carried by the feedback resistor in the headstage, the amount of
supercharging that can be used is limited by saturation of the
current-to-voltage converter in the headstage.
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A brief charging pulse is added to the start and the end of Vcmd.
The size and duration of the charging pulse is empirically
determined to give the fastest charging current. Vm charges
rapidly to its final value. Vcmd and the supercharging pulses are
not part of a positive feedback circuit.
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Fast
Response to
Vcmd Change
Fast
Response to
Rm Change
Stability
Correction
Yes
Yes
Yes
Not guaranteed
Prediction
No
Yes
No
Guaranteed
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Pipette-Capacitance Compensation
When the command voltage is stepped, a large amount of current flows
into the pipette capacitance during the transition from one potential to
the next. In an intracellular micropipette amplifier, such as the
Axoclamp 900A amplifier, this is reduced by setting the capacitance
neutralization controls, as discussed in the earlier section titled
Capacitance Compensation. In a patch-clamp amplifier, such as the
Axopatch 200 and MultiClamp 700 amplifier series, a variation of the
technique is used for the same purposeto reduce or even eliminate
the transient. This section discusses pipette capacitance compensation
in a patch clamp.
Compensating the pipette capacitance in a patch clamp has three
purposes. First, many researchers want to remove the transient from
the records for cosmetic reasons. Second, during the transient the
potential at the top of the pipette is changing slowly while the pipette
capacitance charges. By rapidly charging the pipette capacitance
through the compensation circuitry, the potential at the top of the
pipette is stepped more rapidly, reducing the likelihood that rapid-onset
ionic currents will be distorted. Third, uncompensated pipette
capacitance has a detrimental effect on the stability of the seriesresistance correction circuitry. The component of the current that flows
into the pipette capacitance is not in series with any resistance. Thus
the series-resistance correction circuit exceeds 100% compensation for
this component of the current as soon as the circuit is switched in.
In the Axopatch and MultiClamp amplifiers, two pairs of pipette
capacitance compensation controls are available. With these controls, it
is often possible to reduce the pipette transients to extremely low
levels. The bulk of the transient is reduced by using the fast magnitude
and time constant () controls. The magnitude control compensates
the net charge. The control adjusts the time constant of the charge
compensation to match the time constant of the command pathway and
to compensate for small non-idealities in the frequency response of the
pipette and electronics.
The residual slow component seen in many pipettes is reduced by using
the slow magnitude and controls. A simplified circuit of the fast and
slow compensation circuitry is shown in Figure 3-18.
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Absolute Value
The absolute value of the membrane capacitance is displayed on the
whole-cell capacitance dial after the whole-cell current transient has
been eliminated. This value may be used to estimate the surface area
of the cell assuming that the membrane capacitance per unit area is
1 F/cm2.
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Parts of this section have been reprinted with permission from Finkel, A. S.,
Progress in Instrumentation Technology for Recording from Single Channels and
Small Cells, in Cellular and Molecular Neurobiology: A Practical Approach,
Oxford University Press, 1991.
99
membrane patch will leak out through the seal and will not be
measured. The lower the seal resistance the larger the fraction of
undetected current.
Secondly, thermal movement of the charges in the conducting
pathways of the seal constitute the major source of noise in the
recording unless the seal resistance is very high (several gigohms or
more). A high seal resistance is a prerequisite of low-noise recordings.
High-resistance seals, often called gigaseals, require that very clean
pipettes be used and that they only be used once. If this is the case,
gigaseals will routinely form after the application of gentle suction.
When good gigaseals are formed, the noise due to the leakage current
is virtually eliminated and other sources of noise remain the dominant
limitations in the resolution of the recording technique. These are the
noise of the electronics, the pipette glass, the input capacitance and the
feedback resistor. Commercial patch clamp amplifiers such as the
Axopatch 200B amplifier have minimized the noise of the electronics
and the feedback element in the headstage. Chapter 4: Microelectrodes
and Micropipettes on page 117 describes the attributes of desirable
glasses and the ways to fabricate low-noise pipettes. It also discusses
the use of Sylgard coatings to minimize pipette capacitance.
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Problems
The special demands of patch clamping lead to complicating factors in
the design of a good headstage.
1. Integrated circuit operational amplifiers do not have the
required combination of ultra-low noise and sub-picoamp bias
currents. Thus the operational amplifier has to be made from a
composite of low-noise FETs6, such as the U430 type, and
conventional operational amplifiers. The voltage noise of the
input FETs leads to noise current being injected into the input
capacitance. If this input capacitance is large, this source of
noise becomes dominant; it is proportional to the product of the
input capacitance and the noise of the input FETs.
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Problems
There are two major problems that make the capacitor feedback
technology difficult to implement. The first problem is that after a
sustained net DC input current the integrator voltage ramps towards
one of the power-supply limits. When this happens, the integrator must
be reset by discharging the capacitor. The frequency of the resets
depends on the size of Cf and the average input current. For example, if
Cf is 1 pF and the input current is 2 pA, the output of the integrator will
ramp at 2 V/s. If the resetting circuitry is triggered when the integrator
voltage exceeds 10 V, resets will occur every five seconds.
The reset itself lasts approximately 50 s. During the reset,
sample and-hold circuits are used to maintain the current output at its
value immediately prior to the start of the reset. If it is acceptable to
lose 0.1% of the data, resets as frequent as every 50 ms would be
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Micropipette Holders
High-quality micropipette holders are crucial to successful
single-channel recording. Two characteristics of the holder are
important. First, the holder must be mechanically stable. When working
with a cell-attached patch, drift of the micropipette is unacceptable.
Clearly, a low-drift micromanipulator is required (as discussed in
Chapter 2: The Laboratory Setup on page 39). But if the holder is loose
in the headstage connector or if the pipette does not seat properly, drift
will occur despite the high quality of the micromanipulator. One
common, exasperating problem associated with loose holders is
movement of the pipette when suction is applied, which either prevents
seal formation or damages the cell. The HL-U holders from Molecular
Devices fit all recent Axon Conventional Electrophysiology headstages
and have been designed to fit snugly into the headstage connector and
to firmly grip the pipette to prevent drift. Second, the holder must not
introduce noise. To assure this:
1. Select the right holder material. When a holder is connected to
the headstage, even before a pipette is connected, the opencircuit noise goes up slightly. The reasons are not understood.
Empirical tests have shown that when used with glass
micropipettes, polycarbonate adds the least noise. The HL-U
holders use polycarbonate for the pipette cap and the body of
the holder. The portion that plugs into the headstage is made of
Teflon. Teflon in this position does not add noise, and it
facilitates smooth insertion and removal.
2. Use a small pin for the electrical connection. Large pins have
more capacitance contributed by the surrounding grounded
surfaces.
3. Do not use a shield. Surrounding the holder with a driven or
grounded shield adds to the input capacitance and thus
increases the noise.
Definitions
Positive Current
The flow of positive ions out of the headstage into the microelectrode
and out of the microelectrode tip into the preparation is termed positive
current.
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Inward Current
Current that flows across the membrane, from the outside surface to
the inside surface, is termed inward current.
Outward Current
Current that flows across the membrane, from the inside surface to the
outside surface, is termed outward current.
Positive Potential
The term positive potential means a positive voltage at the headstage
input with respect to ground.
Transmembrane Potential
The transmembrane potential (Vm) is the potential at the inside of the
cell minus the potential at the outside. This term is applied equally to
the whole-cell membrane and to membrane patches.
Depolarizing / Hyperpolarizing
The resting Vm value of most cells is negative. If a positive current flows
into the cell, Vm initially becomes less negative. For example, Vm might
shift from an initial resting value of -70 mV to a new value of -20 mV.
Since the absolute magnitude of Vm is smaller, the current is said to
depolarize the cell (for example, it reduces the polarizing voltage
across the membrane). This convention is adhered to even if the
current is so large that the absolute magnitude of Vm becomes larger.
For example, a current that causes Vm to shift from -70 mV to +90 mV
is still said to depolarize the cell. Stated simply, depolarization is a
positive shift in Vm. Conversely, hyperpolarization is a negative shift in
Vm.
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Patch Clamp
By design, the patch-clamp command voltage is positive if it increases
the potential inside the micropipette. Whether it is hyperpolarizing or
depolarizing depends upon whether the patch is cell attached, inside
out or outside out. The patch-clamp pipette current is positive if it
flows from the headstage through the tip of the micropipette into the
patch membrane.
Cell-Attached Patch
The membrane patch is attached to the cell. The pipette is connected to
the outside surface of the membrane. A positive command voltage
causes the transmembrane potential to become more negative,
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Inside-Out Patch
The membrane patch is detached from the cell. The surface that was
originally the inside surface is exposed to the bath solution. Now the
potential on the inside surface is 0 mV (bath potential). The pipette is
still connected to the outside surface of the membrane. A positive
command voltage causes the transmembrane potential to become
more negative, therefore it is hyperpolarizing. For example, to
approximate resting membrane conditions of Vm = -70 mV, the
potential inside the pipette must be adjusted to +70 mV. If the
potential inside the pipette is increased from +70 mV to +90 mV, the
transmembrane potential of the patch hyperpolarizes from -70 mV to
-90 mV.
From the example it can be seen that the transmembrane patch
potential is inversely proportional to the command potential. A positive
pipette current flows through the pipette across the patch membrane
from the outside surface to the inside surface. Therefore a positive
current is inward.
Outside-Out Patch
The membrane patch is detached from the cell in such a way that the
surface that was originally the outside surface remains exposed to the
bath solution. The potential on the outside surface is 0 mV (bath
potential). The pipette interior is connected to what was originally the
inside surface of the membrane. A positive command voltage causes
the transmembrane potential to become less negative, therefore it is
depolarizing. For example, to approximate resting membrane
conditions, assuming that Vm = -70 mV, the potential inside the pipette
must be adjusted to -70 mV. If the potential inside the pipette
is then increased from -70 mV to -50 mV, the transmembrane potential
of the patch depolarizes from -70 mV to -50 mV.
The membrane potential is directly proportional to the command
potential. A positive pipette current flows through the pipette across
the patch membrane from the inside surface to the outside surface.
Therefore a positive current is outward.
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Summary
1. Positive current corresponds to:
Cell-attached patch
patch inward current
Inside-out patch
patch inward current
Outside-out patch
patch outward current
Whole-cell voltage clamp
outward membrane current
Whole-cell current clamp
outward membrane current
2. A positive shift in the command potential is:
Cell-attached patch
hyperpolarizing
Inside-out patch
hyperpolarizing
Outside-out patch
depolarizing
Whole-cell voltage clamp
depolarizing
3. The correspondence between the command potential (Vcmd) and
the transmembrane potential (Vm) is:
Cell-attached patch
Vm = RMP Vcmd
Inside-out patch
Vm = Vcmd
Outside-out patch
Vm = Vcmd
Whole-cell voltage clamp
Vm = Vcmd
References
Patch Clamp
Rae, J.L. and Levis, R.A. Patch voltage clamp of lens epitheleal cells:
theory and practice. Molec. Physiol, 6, 115162, 1984.
Hamill, O. P., Marty, A., Neher, E., Sakmann, B., and Sigworth, F.J.
Improved patchclamp techniques for high-resolution current recording
from cells and cell-free membrane patches. Pflugers Archiv., 391,
85100, 1981.
Sakmann, B. and Neher, E., Eds. Single-Channel Recording. New York:
Plenum Press, 1983.
Joshi, C. & Fernandez, J. M. Capacitance measurements: an analysis of
the phase detector technique used to study excocytosis and
endocytosis. Biophys. J. 53, 885892, 1988.
Finkel, A. S., Progress in instrumentation technology for recording from
single channels and small cells, in Cellular and Molecular Neurobiology:
A Practical Approach. Oxford University Press, 1991.
114
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Space-Clamp Considerations
Rall, W. & Segev, I. Space-clamp problems when voltage clamping
branched neurons with intracellular microelectrodes. in Voltage and
Patch Clamping with Microelectrodes. Eds. T. Smith Jr. et al., Baltimore:
Williams & Wilkins, 1985.
Other
Hille, B. Ionic Channels of Excitable Membranes. Sunderland,
Massachusetts: Sinauer Associates. 1984.
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116
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118
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injected into the tube. Since the tube can be placed quite close to the
pipette tip, the new solution can reach the vicinity of the pipette tip
after a short diffusion delay. The procedure of draining excess solution
from the pipette depends on the specific setup. This technique cannot
be applied to standard intracellular microelectrodes; due to their small
tip size, the tube cannot be placed close enough to the tip to allow
changing the tip solution within a reasonable time.
The pipette is mounted in a holder that contains a Ag/AgCl electrode
and a proper connector to plug into the headstage of the amplifier. The
holder must be constructed of a low-noise, inert material, which is
often chosen empirically. Axon Cellular Neuroscience holders are
constructed from polycarbonate, which yields low noise with several
patch-clamp pipettes. HL-U headstages have an electrode well to
accommodate pipettes with an outside diameter 1.01.7 mm. The
HL-1-17 (for the non-U-type CV-type headstages) and HL-2-17 holders
(for the HS-type and VG-type headstages) have an electrode well that
can accommodate pipettes with an outside diameter of 1.51.7 mm.
Pipettes of this size have an internal bore that allows insertion of a 1
mm diameter Ag/AgCl pellet electrode. The HL-1-12 (for the nonU-type CV-type headstages) and HL-2-12 (for the HS-type and VG-type
headstages) holders have a pipette well that can accommodate pipette
sizes of 1.01.2 mm diameter. This pipette size requires an electrode
made of small-gauge Ag wire coated with AgCl. A simple yet effective
coating procedure is to clean the silver wire down to bare metal using
fine sand paper and immerse the cleaned wire in bleach (Clorox) for
2030 minutes, until the wire is uniformly blackened by the Ag/AgCl
coating. Coating of the silver wire must be repeated whenever the AgCl
becomes scraped from the wire. Disruption of the coat can be
minimized either by heat polishing the back of each pipette before
insertion into the holder or by encapsulating the coated portion of the
wire in a perforated Teflon sleeve.
The selection of the glass used for making the electrodes is more
important for patch pipettes than for intracellular micropipettes. Most
intracellular micropipettes are made from Corning #7740 Pyrex glass
(Corning Inc., Corning, NY). However, when used for tiny current
measurements as in patch recordings, this glass is too noisy. The best
patch pipettes are made from glasses with low loss factors (see
Table 4-1). These glasses generate lower noise, and often have lower
capacitances and fewer time constants to compensate.
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glass-coated wire, current is passed through the wire to heat it. The
amount of current required depends on the softening temperature of
the glass from which the pipette is constructed. The hot platinum wire
heats the electrode tip and causes sharp corners to round and rough
surfaces to smooth. This polishing is best done under direct observation
at a magnification of 600x1500x.
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121
Glass
Loss
Factor
7940
0.0038
11.8
3.8
1580
1724
0.0066
13.8
6.6
926
Description
Quartz (fused silica)
Aluminosilicate
7070
25
11.2
4.1
8161
0.50
12.0
8.3
604
Sylgard
0.581
13.0
2.9
7059
0.584
13.1
5.8
844
7760
0.79
9.4
4.5
780
Borosilicate
7040
1.00
9.6
4.8
700
KG-12
1.00
9.9
6.7
632
High lead
1723
1.00
13.5
6.3
910
Aluminosilicate
High lead
#184 Coating compound
Barium-borosilicate
0010
1.07
8.9
6.7
625
High lead
EN-1
1.30
9.0
5.1
716
7720
1.30
8.8
4.7
755
7056
1.50
10.2
5.7
720
3320
1.50
8.6
4.9
780
7050
1.60
8.8
4.9
705
KG-33
2.20
7.9
4.6
827
Kimax borosilicate
7740
2.60
8.1
5.1
820
Pyrex borosilicate
1720
2.70
11.4
7.2
915
Aluminosilicate
N-51A
3.70
7.2
5.9
785
Borosilicate
R-6
5.10
6.6
7.3
700
Soda lime
0080
6.50
6.4
7.2
695
Soda lime
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Table 4-2 shows the chemical constituents of many of the same glasses
listed in Table 4-1. This table may be useful in deciding which glasses
are likely to contain leachable components that might affect channel
currents.
Table 4-2 Chemical Compositions of Different Glasses.
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As seen in Figure 4-1, glasses with the lowest loss factor exhibit the
lowest noise. Furthermore, noise decreases as the wall thickness
increases (compare 7052 with 7052TW, thin walled). Even greater
improvement in noise can be achieved using quartz (fused silica)
pipettes. The noise of pipettes fabricated from Corning 7070 (low loss
electrical) or Corning 1724 (aluminosilicate) glasses could also be
improved if proper techniques of fabricating patch pipettes from these
glasses would be developed. The noise values shown in Figure 4-1 are
higher than would be obtained with the Axopatch 200B amplifier in
single-channel recording mode since its capacitive headstage produces
lower noise than is produced by standard resistive headstages. It has
been observed that as the noise of the headstage electronics
decreases, the noise of other components, such as holder and glass, is
reduced as well.
Leachable Components
Glasses are complicated substances made of many different
compounds as shown in Table 4-2. While glasses have major
constituents that lead to their classification as soda lime,
aluminosilicate, borosilicate, for example, they have many trace
compounds whose location in the glass is unknown. Glasses may have
components on their surfaces that can leach into an aqueous
environment with which they are in contact. Leachable components
could be particularly problematic in single-channel and whole-cell
recording due to the close proximity of the channels to the glass. The
literature contains reports of undesirable effects of the leaching of
constituents from the glass on membrane currents.
Further Reading
Cota, G., Armstrong, C. M. Potassium channel inactivation induced by
soft-glass patch pipettes. Methods in Enzymology. Plenum Press. New
York and London, 1983.
Sakmann, B., Neher, E. Geometric parameters of pipettes and
membrane patches. Single- Channel Recording. Sakmann, B., Neher,
E., Eds. pp. 3751. Plenum Press. New York and London, 1983.
Cota, G., Armstrong, C. M. Potassium channel inactivation induced by
soft-glass patch pipettes. Biophys. J. 53, 107109, 1988.
Corey, D. P., Stevens, C. F. Science and technology of patch-recording
electrodes. Single-Channel Recording. Sakmann, B., Neher, E. Eds. pp.
5368. Plenum Press, New York and London, 1983.
Hammill, O.P., Marty, A., Neher, E., Sakmann, B., Sigworth, F.J.
Improved patch-clamp techniques for high resolution current-recording
from cells and cell-free membrane patches. Pflugers Arch. 391,
85100, 1981.
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130
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completely strip off the layer by immersing the oocyte for 13 hours in
a calcium-free saline containing 2 mg/mL collagenase Type IA (Sigma
Chemical Company, St. Louis, MO) until about half of the oocytes have
been released from the ovarian lobe; or 2) a less extensive collagenase
treatment followed by manual removal of the follicular layer with
watchmakers forceps. The latter is preferred, since oocytes that
undergo the extensive collagenase treatment sometimes do not survive
as long as those treated less extensively. Some of the problems
introduced by the follicle cell layer are rather trivial. For instance, it is
more difficult to impale a follicle-encased oocyte with an injection
needle or a microelectrode. Other complications, however, can create
major problems. The electrical coupling between the oocyte proper and
the follicle cells means that one records from both. Numerous examples
of oocyte endogenous receptors that turned out to be in the follicle
cell layer were published. In fact, the Xenopus oocyte is a nearly ideal
expression system for ion channels and receptors since it has very few
types of endogenous channels and receptors, an advantage that is
defeated if the follicle cells are left on the oocyte.
One of the striking features of a Xenopus oocyte is its two-toned color
scheme: the animal pole hemisphere is dark colored, while the vegetal
pole is light colored. This polarity is maintained inside the oocyte: the
nucleus is found in the animal pole, and different populations of mRNAs
exist in the hemispheres. In addition, a standing Cl- current flows from
one pole to the other, indicating that the distribution of channels in the
oocyte membrane is not homogeneous. Published reports showed that
ACh receptors expressed from exogenous mRNAs are also unevenly
distributed; although receptors were expressed all over the oocyte,
more receptors were expressed on the vegetal pole. It is not clear if
there is any relation between the site of RNA injection and the location
of the expressed channels on the oocyte membrane. This
non-homogeneity is not important for whole-cell recording, but could
be important for single-channel recording.
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132
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133
Further Reading
Barnard, E.A., Miledi, R., Sumikawa, K. Translation of exogenous
messenger RNA coding for nicotinic acetylcholine receptors induces
functional receptor in Xenopus oocytes. Proc. R. Soc. Lond. B
215:241246, 1982.
Gurdon, J.B., Lane, C.D., Woodland, H.R., Marbaix, G. Use of frog eggs
and oocytes for the study of messenger RNA and its translation in living
cells. Nature. 233:177182, 1971.
Krafte D., Lester, H.A. Expression of functional sodium channels in
stage IIIII Xenopus oocytes. J. Neurosci. Meth. 26:211215, 1989.
Methfessel, C., Witzemann, V., Takahashi, T., Mishina, M., Numa, S.,
Sakmann, B. Patch clamp measurements on Xenopus laevis oocytes:
currents through endogenous channels and implanted acetylcholine
receptor and sodium channels. Pflugers Arch. 407:577588, 1986.
Snutch, T.P. The use of Xenopus oocytes to probe synaptic
communication. Trend Neurosci. 11:250256, 1988.
Stuhmer, W., Methfessel, C., Sakmann, B., Noda, M. Y., Numa, S. Patch
clamp characterization of sodium channels expressed from rat brain
cDNA. Eur. Biophys. J. 14:131138, 1987.
Stuhmer, W., and Parekh, A.B., Electrophysiological Recordings from
Xenopus Oocytes in Single-Channel Recording, 2nd Ed., Sakmann, B.,
and Neher, E., eds., pp. 341355, 1995.
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135
136
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138
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At first, this procedure may take a long time. It is almost inevitable that
an inexperienced experimenter, who has not yet developed a feel for
the tissue, will kill cells by applying pressure or suction that is too
vigorous. With practice, however, one can clean even deep cells quite
rapidly. Careful and repeated adjustment of the focus aids greatly in
determining the degree of cleaning required at each stage. Depending
on the particular type of tissue, a few tricks to facilitate and accelerate
the process of producing clean cells may be applied. For example, in
regions of densely packed somata, such as the CA1 pyramidal cell layer
of the hippocampus, it is possible to remove tissue at the slice surface
over a relatively large length of the cell layer by applying continuous
suction and moving the pipette along the layer as if using a vacuum
cleaner to remove dead tissue. Inspection of the underlying neurons
often reveals at least one clean pyramidal cell.
After completing the cleaning procedure, patch-clamp recordings of
cleaned cells in slices are carried out following essentially the same
procedures that would be applied to cells in culture. All four major
patch-recording configurations (cell-attached, inside-out, outside-out
and whole-cell) are possible, as well as the nystatin-perforated patch
technique (see Recording from Perforated Patches and Perforated
Vesicles on page 154).
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139
Figure 5-3 The Blind technique. Obtaining a recording using the blind
technique by advancing the patch pipette through a tissue slice.
According to the method of Blanton et al., (1989).
Although this technique can be applied successfully, achieving a good
recording on nearly every attempt, it is almost inevitable that at some
point one will encounter some difficulty either in generating seals or
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Blind Technique
Need upright microscope and water Need no special equipment beyond that
immersion objective.
for intracellular recording in slices.
Vibrating microtome almost
essential.
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141
Further Reading
Blanton, M.G., Loturco, J.J., Kriegstein, A.R., Whole cell recording from
neurons in slices of reptilian and mammalian cerebral cortex. J.
Neurosci. Methods. 30:203210, 1989.
Coleman, P.A., Miller, R.F., Measurements of passive membrane
parameters with wholecell recording from neurons in the intact
amphibian retina. J. Neurophysiol. 61:218230, 1989.
Edwards, F.A., Konnerth, A., Sakmann, B., Takahashi, T., A thin slice
preparation for patch clamp recordings from neurones of the
mammalian central nervous system. Pflugers Arch. 414:600612,
1989.
Gahwiler, B.H., Organotypic cultures of neural tissue. Trends Neurosci.
11:484489, 1988.
Hestrin, S., Nicoll, R.A., Perkel, D.J., Sah, P., Analysis of excitatory
synaptic action in the rat hippocampus using whole cell recording from
thin slices. J. Physiol., 422, 203225, 1990.
Rall, W., Segev, I., Space clamp problems when voltage clamping
branched neurons with intracellular microelectrodes. T.G.J. Smith, H.
Lecar, S.J. Redman, P.W. Gage (Eds.), Voltage and Patch Clamping With
Microelectrodes. Bethesda: American Physiological Society, pp.
191215, 1985.
Sakmann, B., Edwards, F., Konnerth, A., Takahashi, T., Patch clamp
techniques used for studying synaptic transmission in slices of
mammalian brain. Q J Exp Physiol 74: 110718, 1989.
Sakmann, B., and Stuart, G., Patch-pipette Recordings from the Soma,
Dendrites, and Axon of Neurons in Brain Slices in Single-Channel
Recording, 2nd Ed., Sakmann, B., and Neher, E., eds. pp. 199211,
1995.
142
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143
144
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145
current steps. After contacting the cell, gentle suction is applied until
the sum of the seal resistance (Rsl) and the axial resistance of the
pipette (Rp) increases by at least 10 fold; increases greater than
100-fold are common. The suction is released to avoid pulling a finger
of membrane and cytoplasm into the pipette since this would increase
the series resistance and lead to errors in voltage control and current
measurement. Since the lumen of the loose-patch pipette is held at the
bath potential and Rp is low compared to Rsl, there is no need to
compensate for the finite value of Rsl.
This method allows one to simultaneously measure whole-cell and
patch currents as well as whole-cell and patch capacitances. The areas
of the cell and of the patch can be estimated by measuring the
whole-cell and patch capacitances. A convenient way to do this is to
apply a triangle-wave voltage command to the whole-cell voltage clamp
and record the resulting whole-cell and patch current waveforms. The
patch capacitance (c) is calculated from the change in the time
derivative of the voltage (dV/dt) at a vertex of the triangle wave and
the corresponding jump in patch current (I), using the following
equation (Neher, 1971; Palti, 1971):
Because the voltage of the patch pipette is equal to the bath voltage
and only the voltage across the membrane patch covered by the
pipette is changing, the capacitance of the patch pipette itself is
rejected. The capacitance of the whole cell is measured in a similar
fashion. However, the interpretation is complicated by current
contributed from poorly clamped regions (see Johnson and Thompson
(1989) for further discussion).
There are several issues to consider when applying this combined
voltage-clamp method. The most important requirement is that the
patch current must be measured from a well-clamped region of
membrane, for example, from a region that is space clamped by the
whole-cell voltage clamp amplifier. In order to accurately measure
current-voltage relationships and limit the capacitive current flowing
across the patch during voltage steps, the loose-patch pipette should
be positioned close to the point where the whole-cell voltage is
controlled. Cell geometry and electrode positioning should be
considered when designing the physical layout of the experiment.
These considerations limit the kinds of preparations that can be studied
with the method. It is reasonable to use the loose-patch pipette to
record local current densities at different points on a well-clamped
spherical cell body. However, it would be inappropriate to record patch
currents from a thin dendrite when the voltage clamp is applied to the
soma.
The most significant error results from current flowing in the seal
resistance between the loose-patch pipette and the bath. This error
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where Rp is the resistance of the loose-patch pipette and Rsl is the seal
resistance. If (Rp + Rsl) is ten times larger than Rp the error in the
measured current is 10%. To minimize the error, one can reduce Rp by
using a patch pipette with a blunt taper and increase Rsl by using
suction to improve the seal with the membrane. Enzyme treatment
with dispase, pronase or trypsin can be used to clean the cell surface
and improve the seal resistance.
Two kinds of errors caused by series resistance should be considered:
1. The voltage drop that results from macroscopic patch currents
flowing through the axial resistance of the patch pipette (Rp) will
produce an insignificant error in most cases. For example, if the
value of Rp is 300 k and the maximal patch current is 500 pA,
the resulting voltage error is 0.15 mV.
2. A more troublesome error occurs when the bath voltage outside
the patch pipette changes due to large whole-cell currents
flowing in the series resistance (Rsr) between the cell membrane
and the bath ground or virtual ground.
The error current is given by:
where Rsr is the sum of the series resistances of the bath, the agar
bridges and the liquidsilver chloride junction at the bath electrode; it
typically has a value of several kilohms.
Bath series resistance creates an insidious problem when membrane
currents are large. However, the error can be reduced by measuring the
bath voltage with a separate electrode and using it as the reference
voltage at the patch-clamp amplifier. Good practice dictates that the
bath voltage be used to correct the membrane voltage signal applied to
the whole-cell clamp amplifier as well. Instability can result, however, if
the bath voltage is measured with a greater bandwidth than the
membrane voltage. To remedy this, one can limit the frequency
response of the bath voltage follower, but this degrades performance
and causes other errors. A better solution is to eliminate the need to
subtract the bath voltage at the other circuits by voltage clamping the
bath to ground potential. This can be accomplished using an
independent voltage-clamp amplifier operating at a high gain and
bandwidth. Two separate agar bridges and silver-chloride electrodes
can be used to measure the bath voltage and pass current to the bath.
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147
Because of the low resistance of these electrodes, a simple voltageclamp circuit employing a single operational amplifier is adequate for
clamping the bath to ground potential. This arrangement ensures that
the bath voltage does not change during clamp steps. The output of the
circuit provides the record of whole-cell current.
Other problems can be overcome by a careful design of the
experimental layout. Since this method uses several voltage-clamp
amplifiers working simultaneously, it is necessary to limit capacitive
coupling between electrodes. This can be achieved by inserting a
grounded shield between the whole-cell current electrode and the
macropatch pipette, and by lowering the volume of saline to the
minimum required for covering the cell. To reduce high frequency noise
in the patch recording, the submerged tip of the pipette should be
coated with Sylgard (Dow Corning, Midland, MI) and only the tip should
be filled with saline. Johnson and Thompson (1989) provide a thorough
analysis of the problems and the approaches used to reduce errors to
acceptable levels.
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149
guard. Since the inner and outer barrels are at the same potential
during the command steps, current loss from the central membrane
region to ground is minimized and Rsl takes a value that is effectively
infinite. This approach is historically related to vaseline-gap and
sucrose-gap voltage-clamp methods. It has been used successfully to
voltage clamp mammalian skeletal muscle. However, the
implementation is somewhat difficult since it requires the construction
of an elaborate concentric electrode and some specialized
instrumentation. It may still be the best approach in some cases and
the original literature should be consulted for further details.
References
Almers, W., Stanfield, P.R., Stuhmer, W., Lateral distribution of sodium
and potassium channels in frog skeletal muscle: measurements with a
patch-clamp technique. J.Physiol. 336, 261284, 1983a.
Almers, W., Stanfield, P.R., Stuhmer, W., Slow changes in currents
through sodium channels in frog muscle membrane. J. Physiol. 339,
253271, 1983b.
Almers, W., Roberts, W.M., Ruff, R.L., Voltage clamp of rat and human
skeletal muscle: measurements with an improved loose-patch
technique. J.Physiol. 347, 751768, 1984.
Hoshi, T., Zagotta, W.N., Aldrich R.W., Biophysical and molecular
mechanisms of Shaker potassium channel inactivation. Science 250,
533538, 1990.
Johnson, J.W, Thompson, S., Measurement of non-uniform current
density and current kinetics in Aplysia neurons using a large patch
method. Biophys. J. 55, 299308, 1989.
Milton, R.L., Caldwell, J.H., Na current in membrane blebs: Implications
for channel mobility and patch clamp recording. J. Neurosci. 10,
885893, 1990.
Neher, E., Two fast transient current components during voltage clamp
on snail neurons. J.Gen.Physiol. 58, 3653, 1971.
Neher, E., Lux, H.D., Properties of somatic membrane patches of snail
neurons under voltage clamp. Pflugers Arch. 322, 3538, 1971.
Palti, Y., Varying potential control voltage clamp on axons. in Biophysics
and Physiology of Excitable Membranes. W.F. Adelman, Jr., Ed. Von
Nostrand Reinhold Co. pp. 194205, 1971.
Premack, B.A., Thompson, S., Coombs-Hahn, J., Clustered distribution
and variability in kinetics of transient K channels in molluscan neuron
cell bodies. J. Neurosci. 9, 40894099, 1990.
Stuhmer, W., Roberts, W.M., Almers, W., The loose patch clamp. in
Single-Channel Recording. Sakmann, B., Neher, E., Eds. pp. 123132,
1983.
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151
Pipette Fabrication
Large-diameter (1.51.8 mm), thin-walled glass pipettes are used to
fabricate pipettes with large-diameter tips and relatively steep descents
at their tips. The type of glass is unimportant. A conventional
double-pull technique using standard patch-pipette pullers can be
employed. For work with myocytes, light fire polishing with barely
visible effects on the tip favors the spontaneous formation of inside-out
patches upon membrane excision, rather than vesicles that must be
disrupted mechanically. Pipette A in Figure 5-5 shows a typical pipette
tip with a 17 m inner diameter.
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Figure 5-5 Pipette fabrication for giant excised patch method. (contd)
C
Seal Formation
Seals are formed in the usual manner by applying gentle negative
pressure at the cell surface. Maintenance of positive pressure up to the
time of sealing is essential both to keep the electrode tip clean and to
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Further Reading
Collins, A., Somlyo, A. V., Hilgemann, D. W., The giant cardiac
membrane patch method: Simulation of outward Na/Ca exchange
current by MgATP. J. Physiol. 454, 2757, 1992.
Hilgemann, D. W., Giant excised cardiac sarcolemmal membrane
patches: sodium and sodium-calcium exchange currents. Pflugers
Archiv. 415, 247249, 1989.
Hilgemann, D.W., The Giant Membrane Patch in Single-Channel
Recording, 2nd Ed., Sakmann, B., Neher, E., eds. pp. 307326, 1995.
Hilgemann, D. W., Regulation and deregulation of cardiac Na-Ca
exchange in giant excised sarcolemmal membrane patches. Nature
344, 242245, 1990.
Standen, N. B., Stanfield, P. R., Ward, T. A., Wilson, S. W. A new
preparation for recording single-channel currents from skeletal muscle.
Proceedings of the Royal Society of London (Biology) B221, 455464,
1984.
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Further Reading
Falke, L.C., Gillis, K.D., Pressel, D.M., Misler, S., Perforated patch
recording allows longterm monitoring of metabolite-induced electrical
activity and voltage-dependent Ca2+ currents in pancreatic islet B-cells.
FEBS Lett., 251: 167172, 1989.
Finkelstein, A., Water movement through lipid bilayers, pores, and
plasma membranes: theory and reality., Vol. 4, Wiley-Interscience,
New York, p. 127, 1987.
Holz, R., Finkelstein, A., The water and nonelectrolyte permeability
induced in thin lipid membranes by the polyene antibiotics nystatin and
amphotericin B. J. Gen. Physiol., 56: 125145, 1970.
Horn, R., Diffusion of nystatin in plasma membrane is inhibited by a
glass-membrane seal. Biophys. J., 60: 329333, 1991.
Horn, R., Marty, A., Muscarinic activation of ionic currents measured by
a new whole-cell recording method. J. Gen. Physiol., 92: 145159,
1988.
Korn, S.J., Bolden, A., Horn, R., Control of action potential duration and
calcium influx by the calcium-dependent chloride current in AtT-20
pituitary cells. J. Physiol. (Lond), 439: 423437, 1991.
Korn, S.J., Horn, R., Influence of sodium-calcium exchange on calcium
current rundown and the duration of calcium-dependent chloride
currents in pituitary cells, studied with whole cell and perforated patch
recording. J. Gen. Physiol., 94: 789812, 1989.
Korn, S.J., Marty, A., Connor, J.A., Horn, R., Perforated patch recording.
Methods in Neuroscience 4: 264373, 1991.
Kurachi, Y., Asano, Y., Takikawa, R., Sugimoto, T., Cardiac Ca current
does not run down and is very sensitive to isoprenaline in the nystatinmethod of whole cell recording. Archiv. Pharm., 340: 219222, 1990.
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where en is the rms noise voltage (V/ Hz) and Xc is the capacitive
reactance of the bilayer. The capacitive reactance is 1/(2Cbf), where f
is the frequency and Cb is the bilayer capacitance. Therefore:
where Re{Y(f)} is the real part of the admittance of Ra in series with Cb,
and is given by
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component. The width of the annulus (for example, in the plane of the
bilayer) is also roughly proportional to the thickness of the margin of
the aperture. A substantial, further reduction in the electrostrictive
component can be achieved by using the shaved-aperture method,
because it produces a margin of the aperture that is very thin (a few
micrometers). Other techniques, such as drilling or punching apertures,
produce a margin that is much thicker. Finally, any manipulation of the
bilayer that produces a bulkier annulus, such as addition of excess
solvent or repeated wiping, can greatly increase the electrostrictive
component and should be avoided.
Use Capacitance Compensation. The capacitance compensation
circuits in most commercially available patch clamps are only slightly
effective in reducing the duration of saturation because they are
designed to compensate the relatively small capacitance of a patch
pipette rather than the much larger capacitance of a planar bilayer.
Special circuits capable of compensating the larger bilayer capacitance
can be added to the headstage, but they require the injection of
compensating current into the amplifier input through a large capacitor,
which increases the total input capacitance and degrades the noise
performance. Sometimes, the electrostrictive capacitive current can be
partially subtracted by adjustment of a capacitance compensation
circuit with a slow time constant. The BL type of CV-7 headstages (such
as the CV-7B/BL) in use with MultiClamp 700 amplifiers has an
increased fast pipette capacitance compensation range that allows
compensation of up to 300 pF of pipette capacitance.
Decrease the Feedback Resistance. The charging time can be
reduced by decreasing the value of Rf, thereby increasing the rate at
which charge can be delivered to the bilayer. However, the price for this
improvement is a proportional increase in the background thermal
noise currents in Rf and, therefore, a concomitant decrease in usable
bandwidth. Although this may work for large conductance channels, it
is hardly a general solution. A better solution is to include both a large
(50 G) and a small (50 M) feedback resistor in the headstage with
logic-controlled circuitry that can switch between the two. This
approach was taken in the CV-4B headstage for the Axopatch 1 series
of Axon Conventional Electrophysiology amplifiers. During a voltage
step, the bilayer capacitance is rapidly charged through the small Rf.
Immediately after the bilayer capacitance is charged, the feedback
pathway is switched to the large Rf, enabling high resolution recording
of single-channel activity. This approach was used successfully to study
voltage-dependent activation of K+ channels from squid (Wonderlin et
al., 1990; Wonderlin et al., 1991).
Use a Capacitive-Headstage Patch Amplifier. Capacitive-headstage
patch clamps can very rapidly charge the bilayer capacitance with a
maximum instantaneously injected charge of:
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After cooling, the stylus is retracted and the plastic shaved from the
outer surface, leaving a thin-edged aperture where the cut
intersects the depression in the wall of the cup, as in C. See text for
additional details. Reproduced with permission from Wonderlin et
al., 1990.
Briefly, a conical metal stylus is warmed and pressed against the inner
surface of a plastic cup, forming a cone-shaped depression extending
part way across the cup (Figure 5-7). The stylus is then removed and
the plastic shaved away from the outer surface, using a disposable
microtome blade, until the depression is intersected. The size of the
aperture is controlled by varying the depth of shaving. Apertures made
using this method have a thin margin beyond which the plastic rapidly
increases in thickness, thereby decreasing the stray capacitance across
the partition and providing good mechanical strength. The thin margin
of the shaved aperture is very important in reducing the size of the
annulus and, therefore, the electrostrictive change in capacitance
following voltage steps. Following are the essential steps in
implementing the method:
1. Making the stylus. The key to successfully using the shavedaperture method is the manufacture of a high-quality stylus.
Using a lathe and dissecting microscope, stainless steel darning
needles can be ground to a very fine tip (< 5 m diameter) and
polished to a very smooth finish using ultra-fine abrasive paper
(Thomas Scientific, Swedesboro, NJ). Softer metals can be
substituted, but it is more difficult to produce the highly polished
tip. The stylus should be examined under high magnification
(400x) to ensure that the tip is very smooth.
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2. Selecting the plastic. The original description of the shavedaperture technique (Wonderlin et al., 1990) recommended
polystyrene as a good plastic for recording cups. Since that
time, repeated difficulties have been encountered with crazing
of the margin of apertures made in polystyrene cups, which
sometimes appears to make the cups electrically leaky. More
recently, shaved apertures in cups were made from Ultra-Clear
centrifuge tubes (Beckman, Colombia, MD). Also, if a flat
partition rather than a cup is preferred, shaved apertures have
been formed in plastic coverslips (Fisher Scientific, Pittsburgh,
PA). The margin of these apertures does not craze and bilayers
formed on apertures shaved in these plastics are very stable,
lasting several hours. Other plastics can probably be
substituted, with the basic requirement being that they cut
cleanly so that the margin of the aperture is very smooth.
3. Melting the plastic. There are many choices of mechanical
apparatus for manipulating and heating the stylus. A rather
simple method is to mount the stylus in a hole in the tip of a
variable temperature soldering iron, and then to mount the
soldering iron on a manipulator so that the tip can be carefully
pushed into the plastic. Control of the heat is important to
ensure replicability and to avoid overheating, which may
damage the plastic and, perhaps, the stylus.
4. Shaving the aperture. It is easiest to shave the apertures
while observing with a dissecting microscope. The shaved
apertures should be examined under high magnification (400x)
before use to ensure they are free from deformations that might
interfere with bilayer stability.
Viewing with a Microscope
It is not practical to attempt to work with small bilayers without
observing them with a microscope. Bilayers are usually observed under
reflected light because the disappearance of reflected light provides a
good monitor of bilayer thinning. Quite often, the bilayer is viewed
through one eyepiece of a dissecting microscope, while the bilayer is
illuminated by light focused from a fiber bundle through the second
eyepiece. This method is not useful for small bilayers because of the
lack of detail visible with reflected light and the relatively low
magnification (4080x) of most dissecting microscopes. Using a
horizontally mounted compound microscope (Nikon Inc., Garden City,
NY) with a long working-distance objective (Nikon, E-Plan 10x) and a
bilayer recording chamber designed for transillumination provides very
good resolution of detail in the bilayer and annulus at a magnification of
100x.
Testing the System
The performance of the recording system should be tested using a
dummy bilayer, especially if voltage steps are to be used. The dummy
bilayer should use a capacitor with a value close to that expected for
the bilayer capacitance. It may also be useful to add a resistor in series
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Current record from which the capacitive current and switching artifact
have been digitally subtracted. Blank traces (7 traces in which the
channel was not active) were averaged and subtracted from the active
trace. Subtraction is very effective in removing the step artifact, and
high-resolution recording is established within 1ms after the voltage
step.
Logic pulse used to switch the headstage gain. The headstage was
switched to low gain during the rectangular pulse. Reproduced with
permission from Wonderlin et al., 1990.
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Further Reading
Wonderlin, W.F., Finkel, A., French, R.J., Optimizing planar lipid bilayer
single-channel recordings for high resolution with rapid voltage steps.
Biophys. J., 58: 289297, 1990.
Wonderlin, W.F., French, R.J., Ion channels in transit: voltage-gated Na
and K channels in axoplasmic organelles of the squid Loligo pealei.
Proc. Natl. Acad. Sci. USA, 88: 43914395, 1991.
Hamill, O.P., Marty, A., Neher, E., Sakmann, B., Sigworth, F.J. Improved
patch-clamp techniques for high-resolution current recording from cells
and cell-free membrane patches. Pflugers Arch. Eur. J. Physiol.
391:85100, 1981.
Sigworth, F.J., Electronic design of the patch clamp. in Single-Channel
Recording, 2nd Edition, pp. 95126, Sakmann, B., Neher, E. Eds.
Plenum Press, New York, 1995.
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Fundamentals of Filtering
Filters are distinguished by a number of important features. These are:
-3 dB Frequency
The -3 dB frequency (f-3) is the frequency at which the signal voltage at
the output of the filter falls to 1/2, for example, 0.7071, of the
amplitude of the input signal. Equivalently, f-3 is the frequency at which
the signal power at the output of the filter falls to half of the power of
the input signal. (See Decibels (dB) on page 184.)
Order
A simple filter made from one resistor and one capacitor is known as a
first-order filter. Electrical engineers call it a single-pole filter. Each
capacitor in an active filter is usually associated with one pole. The
higher the order of a filter, the more completely out-of-band signals are
rejected. In a first-order filter, the attenuation of signals above f-3
increases at 6 dB/octave, which is equal to 20 dB/decade. In linear
terminology, this attenuation rate can be re-stated as a voltage
attenuation increasing by 2 for each doubling of frequency, or by 10 for
each ten-fold increase in frequency.
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Filter Function
There are many possible transfer functions that can be implemented by
active filters. The most common filters are: Elliptic, Cauer, Chebyshev,
Bessel and Butterworth. The frequency responses of the latter two are
illustrated in Figure 6-1. Any of these filter transfer functions can be
adapted to implement a high-pass, low-pass, band-pass or band-reject
filter. All of these filter transfer functions and more can be implemented
by digital filter algorithms. Digital filters can even do the seemingly
impossible: since all of the data may be present when the filtering
begins, some digital filters use data that arrive after the current
data. This is clearly impossible in an analog filter because the future
cannot be predicted. Typical digital filters are the box-car smoothing
filter and the Gaussian filter.
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Filter Terminology
The terminology in this section is defined and illustrated in terms of a
low-pass filter. The definitions can easily be modified to describe
high-pass and band-pass filters. Many of these terms are illustrated in
Figure 6-2.
- 3 dB Frequency
f-3, defined previously, is sometimes called the cutoff frequency or the
corner frequency. While most engineers and physiologists specify a
filters bandwidth in terms of the -3 dB frequency, for obscure reasons
some manufacturers label the filter frequency on the front panel of
their instruments based on a frequency calculated from the intersection
frequency of the pass-band and the stop-band asymptotes. This is very
confusing. To make sure that the filter is calibrated in terms of the
-3 dB frequency, a sine wave generator can be used to find the -3 dB
frequency.
Attenuation
Attenuation is the reciprocal of gain. For example, if the gain is 0.1 the
attenuation is 10. The term attenuation is often preferred to gain when
describing the amplitude response of filters since many filters have a
maximum gain of unity. (For accurate measurements, note that even
filters with a stated gain of unity can differ from 1.00 by a few percent.)
Pass Band
Pass band is the frequency region below f-3. In the ideal low-pass filter
there would be no attenuation in the pass band. In practice, the gain of
the filter gradually reduces from unity to 0.7071 (for example, -3 dB)
as the signal frequencies increase from DC to f-3.
Stop Band
Stop band is the frequency region above f-3. In the ideal low-pass filter,
the attenuation of signals in the stop band would be infinite. In
practice, the gain of the filter gradually reduces from 0.7071 to a
filter-function and implementation-dependent minimum as the signal
frequencies increase from f-3 to the maximum frequencies in the
system.
Phase Shift
The phase of sinusoidal components of the input signal is shifted by the
filter. If the phase shift in the pass band is linearly dependent on the
frequency of the sinusoidal components, the distortion of the signal
waveform is minimal.
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Overshoot
When the phase shift in the pass band is not linearly dependent on the
frequency of the sinusoidal component, the filtered signal generally
exhibits overshoot. That is, the response to a step transiently exceeds
the final value.
Octave
An octave is a range of frequencies where the largest frequency is
double the lowest frequency.
Decade
A decade is a range of frequencies where the largest frequency is ten
times the lowest frequency.
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Decibels (dB)
Since filters span many orders of magnitude of frequency and
amplitude, it is common to describe filter characteristics using
logarithmic terminology. Decibels provide the means of stating ratios of
two voltages or two powers.
Voltage:
184
Voltage Ratio
Power Ratio
3 dB
1.414:1
2:1
6 dB
2:1
4:1
20 dB
10:1
100:1
40 dB
100:1
10,000:1
60 dB
1,000:1
1,000,000:1
66 dB
2,000:1
4,000,000:1
72 dB
4,000:1
16,000,000:1
80 dB
10,000:1
108:1
100 dB
100,000:1
1010:1
120 dB
1,000,000:1
1012:1
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Order
As mentioned above, the order of a filter describes the number of
poles. The order is often described as the slope of the attenuation in
the stop band, well above f-3, so that the slope of the attenuation has
approached its asymptotic value (Figure 6-2). Each row in the following
table contains different descriptions of the same order filter.
Pole
Order
1 pole
1st order
2 poles
2nd order
Slopes
6 dB/octave
20 dB/decade
12 dB/octave
40 dB/decade
4 poles
4th order
24 dB/octave
80 dB/decade
8 poles
8th order
48 dB/octave
160 dB/decade
Typically, the higher the order of the filter, the less the attenuation in
the pass band. That is, the slope of the filter in the pass band is flatter
for higher order filters (Figure 6-3).
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Shows an inappropriate use of the notch filter. The notch filter is tuned
for 50 Hz. The input to the notch filter is a 10 ms wide pulse. This pulse
has a strong component at 50 Hz that is almost eliminated by the notch
filter. Thus, the output is grossly distorted.
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Sampling Rate
If one intends to keep the data in the time domain, sufficient samples
must be taken so that transients and pulses are adequately sampled.
The Nyquist Sampling Theorem states that a bare minimum sampling
rate is twice the signal bandwidth; that is, the -3 dB frequency of the
low-pass filter must be set at one half the sampling rate or lower.
Therefore, if the filter -3 dB frequency is 1 kHz, the sampling theorem
requires a minimum sampling rate of 2 kHz. In practice, a significantly
higher sampling rate is often used because it is not practical to
implement the reconstruction filters that would be required to
reconstruct time-domain data acquired at the minimum sampling rate.
A sampling rate of five or more times the -3 dB frequency of the filter is
common.
If you wish to make peak-to-peak measurements of your data for
high-frequency signals, you must consider the sampling rate closely.
The largest errors occur when samples are equally spaced around the
true peak in the record. This gives the worst estimate of the peak
value. To illustrate the magnitude of this problem, assume that the
signal is a sine wave and that the following number of samples are
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taken per cycle of the sine wave. The maximum errors that one could
get are then as follows:
5 samples/cycle
19.0% error
10 samples/cycle
4.9% error
20 samples/cycle
1.2% error
The sampling of the peak values varies between no error and the
maximum as stated above.
If one intends to transform the data into the frequency domain,
Butterworth or Elliptic filters are more suitable than the Bessel filter.
These filters have a sharper cutoff near the -3 dB frequency than the
Bessel filter, and thus better prevent the phenomenon known as
aliasing. With a fourth-order or higher Butterworth filter, it is usual to
set f-3 to about 40% of the sampling rate. Frequently, researchers do
not have a Butterworth filter handy. If a Bessel filter is available, it can
be used instead, but f-3 would normally be set to about 2530% of the
sampling rate. This is because the Bessel filter attenuation is not as
sharp near the -3 dB frequency as that of a Butterworth filter.
4-Pole
Bessel
8-Pole
Bessel
4-Pole
Butterworth
8-Pole
Butterworth
1 kHz
1.0
0.97
0.95
0.93
5 kHz
1.0
0.97
0.85
0.82
10 kHz
1.0
0.94
0.83
0.80
The table shows that the main reduction in noise is gained by using a
Butterworth filter instead of a Bessel filter. The improvement achieved
by going from a 4-pole filter to an 8-pole filter of the same kind is
small.
However, for the reasons previously discussed above, the Butterworth
filter cannot be used for time-domain analysis. Since this is the most
common kind of analysis performed on patch-clamp data, Bessel filters
are almost invariably used.
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Digital Filters
Some researchers prefer to record data at wide bandwidths to prevent
the loss of potentially important information. An analog filter is used to
provide anti-alias filtering, followed by a digital filter implemented at
various lower -3 dB frequencies during the analysis. There are many
types of digital filters. Two types are described here:
Nonrecursive Filter
The output of a nonrecursive filter depends only on the input data.
There is no dependence on the history of previous outputs. An example
is the smoothing-by-3s filter:
where yn and xn are the output and input samples at sample interval n.
Nonrecursive filters are also known as finite impulse response filters
(FIR) because their response to a single impulse endures only as long
as the newest sample included in the formula (for example, xn+1 in the
smoothing-by-3s filter).
Another example of a nonrecursive digital filter is the Gaussian filter. It
has a similar form to the smoothing-by-3s filter described above,
except that typically there are more terms and the magnitudes of the
coefficients lie on the bell-shaped Gaussian curve.
These types of filters have the advantage of not altering the phase of
the signal. That is, the mid-point for the rise time of a step occurs at
the same time both before and after filtering. In contrast, analog filters
always introduce a delay into the filtered signal.
A problem with digital filters is that values near the beginning and end
of the data cannot be properly computed. For example, in the formula
above, if the sample is the first point in the data, xn-1 does not exist.
This may not be a problem for a long sequence of data points; however,
the end effects can be serious for a short sequence. There is no good
solution other than to use short filters (for example, few terms). Adding
values outside the sequence of data is arbitrary and can lead to
misleading results.
Recursive Filter
The output of a recursive filter depends not only on the inputs, but on
the previous outputs as well. That is, the filter has some
time-dependent memory. Recursive filters are also known as infinite
impulse response filters (IIR) because their response to a single
impulse extends indefinitely into the future (subject to computer
processing limitations).
Digital-filter implementations of analog filters such as the Bessel,
Butterworth, and RC filters are recursive.
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Where to Amplify
Amplification is possible at a number of different points along the signal
pathway. Since the amplification, filtering and offset circuits can
themselves introduce noise, the location of the amplification circuitry
must be carefully considered. There are several options:
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Offset Control
In some cases, it is necessary to add an offset to the signal before it is
amplified. This is necessary if the gain required to amplify the signal of
interest would amplify the DC offset of the signal to the point that it
would cause the gain amplifier to saturate.
An example is provided by an electronic thermometer. A typical
sensitivity of an electronic thermometer is 10 mV/C, with zero volts
corresponding to 0C. A 12-bit A/D converter can measure with an
approximate resolution of 5 mV, corresponding to 0.5C in this
example. If the data are to be analyzed at 0.01C resolution,
amplification by a factor of at least x50, and probably x100, would be
necessary. If the temperatures of interest are between 30C and 40C
and are amplified by x100, these temperatures will correspond to
voltages between 30 V and 40 V values well beyond the range of the
A/D converter. The solution is to introduce an offset of -350 mV before
any amplification, so that zero volts will correspond to 35C. Now when
the signal is amplified by x100, the 3040C temperature range will
correspond to voltages between -5 V and +5 V.
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Time Constant
The AC-coupling frequency is related to the time constant of decay,
(Figure 6-7):
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(ms)
1.6
5.3
10.0
16.0
3.0
53.0
1.0
160.0
0.1
1,600.0
Saturation
The AC-coupling circuit is the first circuit in most signal conditioning
pathways. If a large step is applied to the AC-coupled inputs, the AC
coupling rejects the step voltage with a time constant determined by
the AC-coupling frequency. If the amplifiers are set for high gain, the
output might be saturated for a considerable time. For example, if the
gain is x100, the AC coupling is 1 Hz and the step amplitude is 1 V, the
output will be saturated until the voltage at the output of the
AC-coupling circuit falls to 100 mV from its initial peak of 1 V. This will
take about 2.3 time constants. Since the time constant is 160 ms, the
output will be saturated for at least 370 ms. For the next several time
constants, the output will settle towards zero.
Overload Detection
An amplified signal may exceed the 10 V acceptable operating range
inside the instrument in two places: at the input of the various filters
and at the output of the final amplifier stage. An example of an
overload condition at the input of an internal low-pass filter is shown in
Figure 6-6.
It is common practice to place overload-detection circuitry inside the
instrument at these points. The overload-detection circuitry is activated
whenever the signal exceeds an upper or lower threshold such as
11 V. The limit of 11 V exceeds the usual 10 V recommended
operating range in order to provide some headroom. There is no difficulty
in providing this headroom since the internal amplifiers in most
instruments operate linearly for signal levels up to about 12 V.
Normally, the overload circuitry simply indicates the overload condition
by flashing a light on the front panel. In more sophisticated instruments
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such as the CyberAmp amplifier, the host computer can interrogate the
CyberAmp amplifier to determine if an overload has occurred.
Averaging
Averaging is a way to increase the signal-to-noise ratio in those cases
where the frequency spectrum of the noise and the signal overlap. In
these cases, conventional filtering does not help because if the -3 dB
frequency of the filter is set to reject the noise, it also rejects the
signal.
Averaging is applicable only to repetitive signals, when many sweeps of
data are collected along with precise timing information to keep track of
the exact moment that the signal commences or crosses a threshold.
All of these sweeps are summed, then divided by the total number of
sweeps (N) to form the average. Before the final division, the amplitude
of the signal in the accumulated total will have increased by N. Because
the noise in each sweep is uncorrelated with the noise in any of the
other sweeps, the amplitude of the noise in the accumulated signal will
only have increased by N. After the division, the signal will have a
magnitude of unity, whereas the noise will have a magnitude of 1/ N.
Thus, the signal-to-noise ratio increases by N.
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Peak-To-Peak
Thresholds
95.0%
99.0%
99.9%
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Blanking
In certain experiments, relatively huge transients are superimposed on
the signal and corrupt the recording. This problem commonly occurs
during extracellular recording of nerve impulses evoked by a
high-voltage stimulator. If the isolation of the stimulator is not perfect
(it never is), there is some coupling of the stimulus into the
micropipette input. This relatively large artifact can sometimes cause
the coupling capacitors in subsequent AC amplifiers to saturate. There
might be some time lost while these capacitors recover from saturation,
and thus valuable data might be wasted.
If it is not possible to prevent the stimulus coupling, the next best thing
to do is to suppress the artifact before it feeds into the AC-coupled
amplifiers. This is made possible in the Axoclamp 900A amplifier by
providing sample-and-hold amplifiers early in the signal pathway. The
user provides a logic-level pulse that encompasses the period of the
transient. This logic-level pulse forces the sample-and-hold amplifiers
into the hold mode. In this mode, signals at the input of the
sample-and-hold amplifier are ignored. Instead, the output of the
sample-and-hold amplifier is kept equal to the signal that existed at the
moment the logic-level pulse was applied.
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Electrode Test
It is useful to be able to measure the electrode resistance for two
reasons. The first reason is to establish the basic continuity of the
electrode circuit. Sometimes, electrode leads can break, leaving an
open-circuit input and, consequently, no incoming data. The second
reason is to verify that the electrode is acceptably attached. For
example, it may be that to achieve the low-noise recording levels
needed in an EMG recording, the electrode resistance must be less than
5 k.
Some transducer amplifiers allow the electrode impedance to be easily
measured. For example, the CyberAmp amplifiers have an Electrode
Test facility. When this is activated, an approximate 1 Ap-p, 10 Hz
square wave is connected to every input via individual 1 M resistors.
The electrode resistance can be directly determined from the amplitude
of the voltage response.
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References
Bell Telephone Laboratories. Transmission Systems for
Communications. By the members of the technical staff at Bell
Telephone Laboratories. Western Electric Company, Inc., Technical
Publications. Winston-Salem, North Carolina, 1970.
Further Reading
Hamming, R. W., Digital Filters. Prentice-Hall, Inc., Englewood Cliffs,
New Jersey, 1977.
Tietze, U., Schenk, Ch., Advanced Electronic Circuits. Springer-Verlag,
Berlin, 1978.
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Thermistors
Thermistors are resistors whose resistance drops significantly as
temperature increases. They are composed of a compressed and
sintered mixture of metallic oxides of manganese, nickel, cobalt,
copper, magnesium, titanium and other metals. Thermistors exhibit
temperature coefficients many times greater than those of pure metals.
For most of them, the resistance falls by 46% per C with increasing
temperature.
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Thermocouples
Thermocouples are economical and rugged transducers, having the
advantage of small size with a very fast response time and a wide
temperature range. Thermocouples consist of two dissimilar metals in
contact. The offset potential between the metals is proportional to
temperature (see Table 7-1). The low sensitivity and broad operating
range generally make thermocouples more suitable for industrial
applications than for physiological ones. Because of their greater
sensitivity, thermistors are more popular than thermocouples for most
physiological applications. On the other hand, thermocouples can be
fabricated in remarkably small sizes, creating the opportunity for some
unusual biological applications. For example, micron-dimension
thermocouples that can be inserted into single living cells have been
described (Cain & Welch, 1974). Commercially available thermocouples
can be obtained with diameters as low as 25 m and thermal time
constants as short as 2 ms (Omega Engineering, Inc., Stamford, CT).
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Transducers
Sensitivity at
Min
Max
Value C Value C 20C V/C
Material
-200
900
60.48
chromel/constantan
-200
750
51.45
iron/constantan
-200
1250
40.28
chromel/alumel
1450
5.80
platinum/Pt-13%
rhodium
1450
5.88
platinum/Pt-10%
rhodium
-200
350
40.28
copper-constantan
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EKG
There are probably more books available on electrocardiogram (EKG)
recording and analysis than any other electrophysiological topic. The
signals are usually of large amplitudes and readily recorded without the
need for any amplifier probes. As electrode spacing is reduced, there is
an increasing possibility of recording unwanted EMG signals from
neighboring muscles. Consequently, the traditional electrode sites are
worth considering. The AI 417 passive 2 mm adapter provides a single
differential EKG channel and plugs directly into the CyberAmp 380
amplifier.
The normal frequency range of the mammalian EKG is 0.2100 Hz; its
amplitude size is up to 23 mV.
EEG
The electroencephalogram (EEG) ranges from 10300 V in amplitude
and has a frequency range from 0.250 Hz. A single differential EEG
channel is best recorded with the assistance of one of the low-noise AI
400 series active probes.
Nerve Cuffs
Nerve-cuff recordings have a frequency response to 10 kHz and an
amplitude in the low microvolt range. The AI 402, x50 Ultra Low-Noise
Differential amplifier probe is designed for this application, with 10 kHz
noise of less than 0.18 Vrms (1.1 Vp-p) in the 0.110 kHz bandwidth.
The nerve cuffs themselves are very simple to make by running bare
stainless steel fine wires through a small length of silicone tubing split
longitudinally. Any bared wires that are in contact with the outer
surface of the cuff can be insulated with silicone adhesive.
Metal Microelectrodes
The impedance of metal microelectrodes may be several hundred
kilohms or more. All of the low-noise AI 400 series active probes are
suitable for recording from high-resistance microelectrodes. Since the
input capacitance of the AI 401 differential amplifier probe is
approximately 5 pF, the largest electrode resistance consistent with
maintaining a 10 kHz bandwidth is about 3 M.
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Transducers
Pressure Measurements
In the past, high-quality pressure transducers were very expensive and
suffered from significant temperature sensitivity. The introduction of
semiconductor pressure transducers has greatly reduced the cost,
decreased the temperature sensitivity and enhanced the range of
transducer devices available.
An important point when measuring very low pressures is to set the
height of the pressure transducer to that of the sense location in order
to avoid hydrostatic errors introduced by fluid-filled catheters. Pressure
transducers should also be kept out of the range of heat sources such
as heat lamps to avoid temperature-induced increases in the signal
output.
Major producers of semiconductor pressure sensors include Honeywell,
Inc. (Minneapolis, MN). Blood pressure transducers from Cobe
Laboratories, Inc. (Lakewood, CO) have a usable range of -50 to
+ 300 mmHg. All pressure transducers can be interfaced to the Axon
CyberAmp 380 amplifier via the AI 490 Connector and AI 491 Cable
Kits.
Force Measurements
The requirements for force measurements are so diverse that many
people need to make their own force transducer, although some are
commercially available (for example, Grass Instruments Company,
Quincy, MA). Force transducers have some compliance and you must
consider this when choosing transducers. For example, with the Grass
Instruments FT10, a force of 100 N will stretch the device by 1 mm.
For user-designed force transducers, the two main choices are between
resistive or semiconductor strain gauges. Resistive gauges are cheaper
and generally more rugged while semiconductor strain gauges are
smaller and about 10 times more sensitive. Strain gauges are available
from Measurement Specialties, Inc. (Hampton, VA).
User-designed force transducers can be interfaced to the CyberAmp
380 amplifier via the AI 490 Connector and AI 491 Cable Kits.
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Acceleration Measurements
Accelerometers can consist of piezoelectric pressure sensors attached
to a test mass, small enough for physiological experiments.
Piezoelectric accelerometers can have a wide frequency range (1 Hz to
25 kHz) and a wide dynamic range (0.012,500 g). Accelerometers can
also consist of strain gauges attached to a mass. Alternative systems
use feedback to prevent the test mass from being displaced; the
amount of the applied feedback force represents the output signal.
Length Measurements
Implantable Length Gauges
Length can be measured inside an animal using mercury-filled or
saline-filled silicone tubing length gauges (Lemon and Prochazka,
1984). As the tubing is stretched, the impedance of the transducer
increases. To avoid the generation of bubbles in the tubing, a
high-frequency AC signal is used with an AC bridge circuit. In practice,
these gauges have many difficulties and may not be much better than
cinematography techniques using markers on the limb joints for
measuring muscle lengths.
Linear Potentiometers
Linear potentiometers are inexpensive and very simple to use but must
be perfectly aligned to avoid internal damage. Linear potentiometers
can be interfaced to the Cyber-Amp 380 amplifier via the AI 490
Connector and AI 491 Cable Kits.
Self-Heating Measurements
As a result of applying an excitation current or voltage to the
temperature measurement sensor, power is dissipated in the sensor.
This power dissipation causes the temperature of the sensor to rise
above the ambient temperature that is being measured. This
phenomenon is referred to as self heating. For most of the
temperature sensors used in physiology, it is necessary to keep the
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Isolation Measurements
In industrial environments, it is common for signal leads from
transducers to be run over long distances past machinery that can
induce large voltages in the leads. To protect the measurement
instrument, isolation amplifiers should be used for each transducer.
Induced voltages are not commonly a problem in animal physiology
applications and, therefore, isolation is not required. Note that if
measurements are to be made from human subjects, isolated probes
that are approved for human use must be used.
Insulation Techniques
Electrodes that are implanted for long-term recording eventually fail
either due to the rupture of a solder joint, breakage of wires at points
of repeated flexion, or moisture penetration through the insulation.
Although teflon-insulated wire is commonly used, when it is connected
to the leads of the transducer it is difficult to join the teflon insulation to
the insulation on the transducer leads. A solution is to etch the teflon so
that it will adhere to the adhesive used to join the two insulations. In
practice it is often faster and better to use PVC-insulated wires because
it is much easier to make a strong adhesion, even though PVC is more
permeable to water over the long term. Also, Araldite AV138M
(CIBAGEIGY) and Epoxylite #6001 (Epoxylite Corporation, Anaheim,
CA) both provide excellent water-resistant insulation for rigid
applications.
Special attention should be paid to providing strain relief where wires,
and particularly connections, are repeatedly flexed. Sliding a length of
silicon tubing over the stress region often reduces this problem.
Further Reading
Cain, C., Welch, A. J., Thin-film temperature sensors for biological
measurement. IEEE Trans. Biomed. Eng. BME-21(5), 421423, 1974.
Cooke, I.R., Brodecky, V., Becker, P.J., Easily implantable electrodes for
chronic recording of electromyogram activity in small fetuses. J.
Neurosci. Meth. 33, 5154, 1990.
Geddes, L. A., Baker, L. E., Principles of Applied Biomedical
Instrumentation. John Wiley & Sons, New York, 1989.
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Horowitz, P., Hill, W., Measurements and signal processing. The Art of
Electronics, 14. Cambridge University Press, Cambridge, 1986.
Lemon, R., Prochazka, A. Eds. Methods for neuronal recording in
conscious animals. IBRO Handbook Series: Methods in the
Neurosciences, Vol. 4. John Wiley & Sons, Chichester, 1984.
Loeb, G. E., Gans, C., Electromyography for Experimentalists.
University of Chicago Press, Chicago, 1986.
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Memory
RAM (Random Access Memory) is the physical device used to store
programs and data while the computer is running. When the computer
power is off, RAM cannot hold any information, so it is referred to as
volatile memory. It is used because instructions and data can be read
from, and written to, RAM much faster than any other type of memory
device. How much RAM you need and can use depends on the type of
computer you buy as well as the capabilities of the programs you use.
The usefulness of extra memory depends on the type of applications
that you will be running. Acquiring and analyzing very large datasets in
general requires more memory than acquiring and analyzing smaller
amounts of data.
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Hard Drive
At the time of writing, 500 GB hard drives are not uncommon, and hard
drives with capacity of a terabyte or more are now widely available to
consumers.
For traditional electrophysiology experiments, these hard drives provide
more than enough storage. For applications that include live cell video
at high frame rates, a terabyte of storage is not enough for long-term
archiving of data. The good news is that hard drives using USB,
Firewire, or SATA can be stacked externally, and are cheap enough to
be able to keep up with the increasing amounts of data.
For data acquisition, hard disk drive performance may be important. To
record long segments of unbroken data, direct acquisition to disk
(rather than memory) is necessary, and the speed of acquisition is
limited by the drive performance. High hard drive speeds of 5,400 RPM
are standard, and for a small price premium 7,200 RPM drives are
readily available.
Graphics Card
Recent versions of the Windows and Macintosh operating systems have
numerous extraneous eye candy features that can consume a large
proportion of a systems resources without any tangible benefit. To free
up the main system processor, dedicated high-powered graphics
processors are now included in most modern computer systems.
Computations required to render high-speed or 3D graphics are
performed on the graphics card, freeing up the system processor for
other tasks. The purchase of a high-end graphics card can therefore
increase overall system performance.
Data Backup
As common and everyday as personal computers have become, hard
disks are complex mechanical devices requiring precise tolerances in
their operation, and are subject to mechanical failure. The lifetime of
hard disks is usually several years, but manufacturing defects, physical
shocks, or too many power-on cycles can make your hard disk ready to
fail at any moment. It is essential that a disk full of programs and data
be backed up regularly.
There are two common methods of backing up data: you can backup to
another hard drive, or back up to an optical medium such as CD or
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Glossary
A/D converter
An Analog-to-Digital converter (ADC) maps analog measurements to
digital numbers that the computer can store and use in calculations.
See Analog and Digital.
Analog
Real signals in the physical world are described as analog, meaning that
the values change in a continuous manner. A mercury thermometer is
an analog measurement device; the mercury rises smoothly in
response to changing temperature. However, the computer requires
discrete digital numbers to describe measurements of the data. See
A/D converter and Digital.
ASCII
American Standard Code for Information Interchange. A standardized
7-bit code for exchanging information between computer systems. The
128-character set consists of the alpha-numeric and punctuation
characters, as well as control codes such as Escape and Carriage
Return. Files on a computer are often stored in a simple ASCII format,
so that they can be easily transferred and read by different systems
and programs.
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Bit
Information is stored in computer memory as a series of bits, which are
binary switches (numbers) that can either be on (1) or off (0). A group
of eight bits is called a byte, which is a common unit for addressing and
storing information.
Boot
To boot a computer refers to the sequence of events that occurs when a
computer is switched on. When the computer is turned on, its built-in
software instructs the computer to load the operating system from disk
into memory, and turns over control of the computer to the operating
system.
Byte
A byte is a series of eight bits of data. Since a bit can have two values
(zero and one), a byte can have 28 (256) values. A character on an IBM
text screen is stored in a byte of memory, so there are 256 distinct
characters available. A byte is a typical unit for storing data and
computer instructions.
Cache
A cache is a place to store often-accessed data or computer
instructions, and is designed to provide quicker access for increased
performance. A memory cache is faster-than-usual RAM for storing
data that the microprocessor requires often from the normal RAM
storage area. A disk cache is a RAM storage area for data that is stored
on disk but is being read frequently.
CPU
Central Processing Unit. See Microprocessor.
D/A converter
A Digital-to-Analog converter (DAC) transforms digital information from
the computer into analog voltages to drive real world devices. See
Analog and Digital.
Data acquisition
In the context of computers, data acquisition refers to the
analog-to-digital conversion of measurements for storage and analysis
by the computer.
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Device driver
Software that acts as an extension to the operating system, usually for
supporting a hardware device. Mouse pointing devices and network
cards are common devices that require a device driver to be loaded
when booting the system up. On PC computers, device drivers are files
that traditionally have a .SYS filename extension.
Digital
Data represented in discrete, discontinuous form is digital, as opposed
to the smooth representation of data as measured by an analog device,
such as a pen chart recorder. Computers require discrete digital
numbers for storage and processing. See A/D converter and Analog.
DPI
Dots Per Inch is a measure of resolution on a printed page or computer
screen. The more dots per inch, the sharper that text or a graphic
image will appear. A laser printer typically has a resolution of 300 DPI.
EEPROM
Electrically Erasable Programmable Read-Only Memory. Allows ROM
memory chips to be reprogrammed with new information by users in
the field. See ROM.
I/O Interfaces
An I/O interface defines the physical interface and communication
method for devices attached to the computer, such as the display,
printer, mouse and disk storage device. For devices attached internally,
such as the display adapter, the physical I/O interface is the computers
internal bus. For external devices, for example, the mouse or printer,
there are several interfaces implemented on Windows and Macintosh
computers.
PARALLEL PORT
The parallel port interface, which exists primarily in the Windows world,
allows eight bits of data to be transferred at a time (in parallel). It
offers high speed, but its design allows mostly one-way
communication; the external device is very limited in the signals it can
send back to the computer. As a result the only devices designed to use
the parallel port are those that mostly receive data, the most common
example being the printer. While most consumer computers implement
this port as a 25-pin connector, commercial printers often use a 36-pin
Centronics connector.
Parallel ports are becoming uncommon with the advent of USB printers.
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SERIAL PORT
The RS-232 serial port is a general communication interface that allows
full, two-way communication, thereby making it much more versatile
than the parallel port. It exists on many types of computer systems.
Common devices which attach to a serial port include the mouse and
the keyboard, but which used dedicated 6-pin PS/2 connectors vs. the
general-use 9-pin DB connector.
Serial ports have already been phased out for consumer devices, but
because communication using the serial interface is relatively
straightforward, serial ports remain useful for low-level device
communication. For device development, therefore, one or two serial
ports is essential.
When buying a new PC computer for use in a laboratory, you should
ensure that it has a serial port.
USB PORT
In just a few years, USB has lived up to its name and become the
Universal Serial Bus. Devices such as cameras, phones, printers,
scanners, external hard drives, flash drives, keyboards, mice and other
consumer products now come with USB as the standard interface. USB
is much faster than older protocols such as serial, and so it is suitable
for applications that require large amounts of data to be transferred at
high speed. Modern scientific instruments such as the MultiClamp
700B and Axoclamp 900A amplifiers are now being made with USB
interfaces, and this trend is likely to continue.
The original implentation of USB 1 has been supplanted by a
high-speed version known as USB 2. USB 2 allows high-speed devices,
such as the Digidata 1440A digitizer, to be easily connected to either
desktop or laptop computers.
KB
A kilobyte is approximately 1,000 bytes. As a computers natural
system of numbers is in base two, a kilobyte actually refers to 210
(1,024) bytes. It is abbreviated with a capital K to distinguish it from
the abbreviation for 1,000, as in kHz.
MB
A megabyte is 1,024 kilobytes, or 1,048,576 bytes. See KB.
Memory
See RAM and ROM.
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Microprocessor
A microprocessor is a single integrated circuit chip that combines
functions for doing logical and mathematical operations, managing
memory and communicating with external devices. It is the main
control center of a microcomputer.
Operating system
An operating system manages the hardware in a computer and
provides a set of services for an application program, such as writing
data to a disk drive.
Parallel port
A type of communication interface that transfers data across several
parallel lines. Communication is mostly one-way, and the length of the
connecting cable is limited. In the PC, the parallel port is used mainly
for connecting printers.
PC
Originally referred to the IBM PC, but now refers to any computer
compatible with the IBM PC designs based upon Intel CPUs.
RAM
Random Access Memory is composed of specialized integrated circuits
designed for storing programs and data. RAM can be quickly read from
and written to, so programs execute quickly.
However, when the computer power is off, RAM cannot hold any
information.
Resolution
The sharpness of images and characters on the computer screen or
on printer output. Most accurately described in units of dots per inch.
ROM
Read Only Memory resides on integrated circuits that hold information
which is not lost when power is turned off. The contents of the ROM are
usually loaded by the manufacturer and are not alterable by the user.
RS-232
A common, standard protocol for communicating over serial ports. It is
a general two-way communication interface that transmits data on a
single line and receives data on a single line.
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SCSI
Small Computer System Interface. This is a type of parallel interface
that provides for higher speeds than were possible in the older serial or
parallel interfaces, while allowing multiple devices to be connected to a
single port.
Serial port
A legacy standard connection, which provides a general two-way
communication interface that transmits data on a single line and
receives data on a single line. The serial interface no longer exists in
most types of computers. Also known as a RS-232 serial port.
Throughput
A measure of performance describing how much data per unit of time
can be transferred from device to device in a computer system.
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Acquisition Hardware
Scientists are always seeking better methods to record and store the
growing amounts of data generated in experiments. In
electrophysiological experiments, the data are most often in the form of
voltage waveforms whose magnitudes vary with time. Data in this form
are appropriate for displaying on an oscilloscope or chart recorder, but
entirely inappropriate for storing on a computer disk. Since computers
can only store discrete numbers, a process of analog-to-digital
conversion must be undertaken to convert the analog data into a
compatible format for the computer.
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Time
Digitized Temperature C
6:00 AM
10
6:30 AM
11
7:00 AM
11
7:30 AM
12
8:00 AM
13
8:30 AM
13
9:00 AM
14
9:30 AM
14
10:00 AM
14
10:30 AM
15
11:00 AM
15
11:30 AM
15
231
Acquisition Hardware
Digitized Temperature C
12:00 PM
17
12:30 PM
18
1:00 PM
19
1:30 PM
19
2:00 PM
19
2:30 PM
18
3:00 PM
16
3:30 PM
15
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Quantization Error
The ADC converter generates binary numbers that have finite
resolution. That is, a small range of analog values will all produce the
same binary number after conversion. Returning to the
temperature-measurement example above, the hypothetical ADC
rounded off all of the temperature values to the nearest degree Celsius
reading. That is, only numbers between 0 and 99C in steps of 1C
were allowed. Clearly, the temperature changes continuously between
each 30-minutes reading, not in 1 C jumps. The range of analog
values that are recorded as the same digital number is the quantization
error.
In an ADC, the total measurement range (for example, 0100C) is
divided into a fixed number of possible values. The number of values is
a power of two, often referred to as the number of bits. Commonly,
these values are:
8 bit = 28 = 256 values
12 bit = 212 = 4,096 values
16 bit = 216 = 65,536 values
To illustrate the impact on the resolution of using an 8-bit, 12-bit or
16-bit ADC, consider the temperature-measurement example where
the electronic thermometer circuit generates an analog output
from-10 V to +10 V for temperatures in the range -100C to +100C.
In this case, the resolutions are:
8 bit = 78.4 mV = 0.784C
12 bit = 4.88 mV = 0.0488C
16 bit = 0.305 mV = 0.00305C
If the ADC is preceded by a programmable-gain amplifier, signals that
occupy only a small range, for example, 1 V, can be amplified before
conversion so that the full resolution of the ADC can be utilized.
The resolution of a 12-bit ADC with an input range of 10 V is 4.88 mV.
Although this resolution does not represent a difficult number for a
computer-based system to use, some researchers preferred a round
number such as 5.00 mV. This could easily be achieved by setting the
span of the ADC to 10.24 V instead of 10 V. Now, many ADC
systems are now designed with the 10.24 V span (for example, the
Axon Digidata 1440A data acquisition system).
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Converter Buzzwords
The terms in this section are defined for a D/A converter. With simple
reorganization, these definitions can be re-cast for an A/D converter.
Only the more esoteric terms have been defined in this section. It is
assumed that the reader already understands conventional terms such
as voltage offset.
Gain Accuracy
Gain accuracy is the closeness with which the output of the D/A
converter corresponds to the specified full-scale value when the digital
input to the D/A converter is the maximum value.
Linearity Error
Linearity error is the maximum deviation of the D/A output from a
straight line drawn between the minimum and the maximum output.
Differential Nonlinearity
When the digital control word changes by a minimal step, for example,
one least-significant bit (LSB), the output value should change by 2-n of
the full-scale value, where n is the number of bits in the converter. Any
deviation from this ideal change is called the differential nonlinearity
error. It is expressed in multiples of LSBs.
Monotonicity
Monotonic behavior means that for every increase in the digital input to
the D/A converter there will be an increase in the analog output. This
requires that the differential nonlinearity error be less than 1 LSB.
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Acquisition Hardware
circuitry into the analog switches. Glitches are worst when a large
number of the bits of the digital input to the D/A are changed. For
example, when the analog output is required to go from negative to
positive, the digital input bits can change from 1111 1111 1111 to 0000
0000 0000. That is, every single bit changes. This change generates
the worst glitch.
Glitches can be prevented by including a sample-and-hold circuit at the
output of the D/A converter. The sample-and-hold is normally in the
sample mode so that the output of the D/A converter feeds straight
through. However, for one or two microseconds, perhaps less, after the
digital input to the D/A converter is updated, hold mode is selected so
that the glitch, if present, is blocked.
In modern integrated-circuit D/A converters, efforts are made in the
design of the chip to match the propagation and activation delays of
each bit in the circuit and to minimize the charge injection so that
glitches are small in the first place. It is thus becoming increasingly less
common for a deglitching circuit to be included. On the other hand, the
high-level of integration in modern D/A converters sometimes
introduces another problem, called feedthrough noise. This problem
occurs when the digital latches that contain the word for the D/A
converter are integrated into the same chip. It may happen that
because of the proximity of the digital circuits to the D/A converter,
digital noise couples into the D/A converter circuit. The coupled signal
manifests itself as a pulse on the output of the D/A converter circuit
each time the digital word is updated. Strangely, this feedthrough noise
often appears even if the D/A value is being held constant. This is
because, for simplicity, most software that simultaneously performs
A/D and D/A conversions updates the D/A word once per A/D sample,
even if the D/A value is not changing. The best solution is to eliminate
the feedthrough noise at its source by using separate integrated circuits
for the D/A converter latches. This is the approach used in the Digidata
1440A digitizer.
Timers
Most ADC systems have several timers available for a multitude of
tasks. The most essential is the provision of a regular clock signal to
initiate each A/D conversion. In most systems, one D/A conversion is
performed for each A/D conversion, but this is not required and some
systems provide for running the D/A converter at a different clock rate
to the A/D converter.
Additional timers are used to implement gear shifting. This is a
technique wherein the acquisition rate is rapidly changed (gear shifted)
during acquisition from a low to a high rate or vice versa, without
stopping the acquisition.
Uncommitted timers are generally provided for use in frequency
measurement, event counting or interval timing. For example, the
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Digital I/O
Digital outputs are used during experiments to control external
equipment. For example, an oscilloscope can be triggered before the
acquisition commences, or a flash lamp or an isolated stimulator could
be activated during the acquisition. In other experiments, several
solenoids can be sequentially activated before, during and after the
acquisition.
Digital inputs are used routinely in industrial control applications for
monitoring the state of solenoids and other apparatuses, but they are
rarely used in electrophysiological experiments. The main application
for digital inputs in electrophysiology is for triggering acquisitions and
for indicating that a tag should be attached to the data.
Optical Isolation
Computers provide quite hostile environments for plug-in data
acquisition boards. The rapid switching activity of the digital logic
circuits causes electronic noise to radiate directly into plug-in boards
installed by the user and to be introduced into the ground reference
used by the plug-in boards.
Nevertheless, it is usually possible to design data acquisition systems
that do not suffer from extraneous noise pickup. This is achieved by
careful layout and good grounding. Grounding and shielding, if applied
with great skill and diligence, can sometimes be made to work
acceptably for the A/D converter, especially when combined with
differential recording. However, it is very difficult to achieve acceptable
noise levels on the DAC outputs. The following approach is often
invoked for 16-bit systems.
First, move the A/D converter off the plug-in board in the computer to a
separate board housed in an external box. This eliminates the problems
due to direct pickup of noise generated inside the computer housing.
However, it does not eliminate the noise introduced through the system
ground. The noise in the system ground can be eliminated by using
optical isolation. In this technique, the digital signals to and from the
computer are not connected directly. Instead, an optical coupler is
interposed in each digital line. A separate power supply is provided for
the A/D and D/A side of the optical couplers. This technique allows a
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Acquisition Hardware
low-noise ground to be maintained for the A/D and D/A converters and
for the experimental setup.
Software Support
Nowadays fewer researchers are writing their own data acquisition and
analysis software using low-level languages and device drivers. Most
researchers are using one of two types of commercially available
software. The first type is a rich development environment such as
MATLAB or LabVIEW. This environment allows researchers to design
their own software without having to become experts in the low-level
control of the data-acquisition hardware. The second type of software is
a turn-key package, such as pCLAMP software. These packages
provide sophisticated acquisition and analysis, but only limited ability to
customize them. Nevertheless, they are extremely popular because
they perform well and are easy to learn.
When choosing hardware and software for a data acquisition system,
we recommend that you choose the software first and then purchase
the hardware recommended by the software applications that you have
selected.
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10
Sampling Rate
The sampling rate used should be selected considering the particular
experiment. Use of excessively high sampling rates wastes disk space
and will increase the time required for analysis. Furthermore, a higher
sampling rate is usually associated with a higher filtering frequency,
which in turn allows a larger amount of noise to contaminate the signal.
Subsequent analysis of the data may therefore require noise reduction
using analysis-time filtering, which can be time-consuming. Guidelines
to choosing the correct sampling rate are discussed in the following
paragraphs (Colquhoun and Sigworth, 1983, and Ogden, 1987).
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Data Analysis
Biological signals are most commonly analyzed in the time domain. This
means that the time dependence of the signals is examined, for
example, to characterize the membrane response to a voltage-clamp
pulse. The usual rule for time-domain signals is that each channel
should be sampled at a frequency between 5 and 10 times its data
bandwidth. Knowing the value of the data bandwidth is required in
order to set the filter cut-off frequency during acquisition and analysis.
For a sinusoidal waveform, the data bandwidth is the frequency of the
sine itself. For most biological signals, the data bandwidth is the highest
frequency of biological information of interest present in the recorded
signal. This can be determined directly by examining a power spectrum
of rapidly sampled unfiltered data, though this is rarely done.
Alternatively, one can estimate the number of points per time interval
required to give a data record whose points can be easily connected
by eye and calculate the sampling rate directly. The data bandwidth and
filter frequency can then be calculated from the sampling rate. For
example, if a fast action potential (1 ms to peak) is to be recorded, 25
samples on the rising phase would yield a reasonably good 40 s
resolution, requiring a sampling rate of 25 kHz and an approximate
data bandwidth of 5 kHz.
The rules are more straightforward in some special cases.
Single-channel recording is discussed below. For signals with
exponential relaxation phases, the sampling rate needed to estimate a
time constant depends on the amount of noise present; for moderately
noise-free data, at least 15 points should be taken per time constant
over a period of 4 to 5 time constants. Many fitting routines will fail if
sampling is performed over only 3 time constants, since the waveform
does not relax sufficiently far towards the baseline. For a sum of
multiple exponentials, the sampling rate is determined in this way from
the fastest phase; sampling must extend to 4 time constants of the
slowest phase. If this would result in too many samples, a split clock
(as in the Clampex program of the Axon pCLAMP suite) or other
methods of slowing the acquisition rate during the acquisition, could be
employed as long as at least 15 points are taken over each time
constant.
When a set of several channels is recorded (for example, channels 0
through 3), most data acquisition systems sample the channels
sequentially rather than simultaneously. This is because the system
usually has only one analog-to-digital converter circuit that must be
shared among the channels in the set. For example, if four channels are
sampled at 10 kHz per channel, one might expect that they would be
sampled simultaneously at 0 s, 100 s, 200 s, etc. Instead, channel
0 is sampled at 0 s, channel 1 at 25 s, channel 2 at 50 s, channel 3
at 75 s, channel 0 again at 100 s, channel 1 again at 125 s, etc.
There is therefore a small time skew between the channels; if this
causes difficulties in analysis or interpretation, a higher sampling rate
can be used to minimize the time skew (but this may cause problems
associated with high sampling rates, as mentioned above).
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Filtering
The signal should be filtered using an analog filter device before it
arrives at the ADC. As discussed in Chapter 6: Signal Conditioning and
Signal Conditioners on page 179 and in Colquhoun and Sigworth, 1983
and Ogden, 1987, this is done to prevent aliasing (folding) of
high-frequency signal and noise components to the lower frequencies
of biological relevance.
Acquisition-time filtering of time-domain signals is usually performed
using a Bessel filter with the cut-off frequency (-3 dB point; see
Chapter 6: Signal Conditioning and Signal Conditioners on page 179)
set to the desired value of the data bandwidth. A 4-pole filter is usually
sufficient unless excessive higher frequency noise requires the 6- or
8-pole version. The Bessel filter minimizes both the overshoot (ringing)
and the dependence of response lag on frequency. The latter two
effects are properties of the Chebyshev and Butterworth filters (see
Chapter 6: Signal Conditioning and Signal Conditioners on page 179
and Chapter 11: Noise in Electrophysiological Measurements on
page 259, or Ogden, 1987), which are less appropriate for time-domain
analysis.
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Data Analysis
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Single-Channel Analysis
Goals and Methods
The goal of single-channel current analysis is to reconstruct the
idealized current waveforms from which information about the
mechanisms of channel function is derived. Specific information can be
deduced with respect to channel state diagrams, kinetics of channel
opening, closing and gating, channel barrier models, and the effects of
channel blocking agents and membrane constituent on channel
function.
Articles that present approaches and methods used in single-channel
current analysis include Colquhoun and Hawkes, 1983; Colquhoun and
Sigworth, 1983; McManus, Blatz and Magleby, 1987; Sigworth and
Sine, 1987; and French et al., 1990.
The current from a single ion channel is idealized as a rectangular
waveform, with a baseline current of zero (closed-channel), an
open-channel current dependent on the conductance and membrane
potential, and rapid transitions between these current levels. In
practice, this idealized current is distorted by the limited bandwidth of
the apparatus and contaminated by noise. Shifts in the baseline may
occur during the recordings, and capacitative current spikes are present
in sweeps in which voltage changes are applied across the membrane.
The current signal is further altered by filtering and by periodic
sampling. These effects can impede making confident inferences from
data regarding channel behavior.
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Data Analysis
Analysis-Time Filtering
The digital Gaussian filter can be used for additional analysis-time
filtering of single-channel records. This introduces symmetrical time
delays at both opening and closing and can therefore be used for the
unbiased estimation of latencies using the 50% criterion (Setting the
Threshold for a Transition on page 244).
Baseline Definition
The accuracy of the threshold method for transition detection depends
on the stability of the baseline (for example, closed-channel) current or,
if the baseline is unstable, on the ability of an automated procedure to
correctly identify the baseline as it changes. A number of ways have
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Missed Events
Events will be missed if their true durations are shorter than the dead
time of the system, which is determined by the filter cut-off frequencies
used during acquisition and analysis. Events will also be missed if their
superthreshold portions happen to miss the times when the signal is
sampled even if their durations are longer than this dead time. The
resulting error is minimal if the fastest time constant in the system is
much longer than the sampling interval because few events will be
shorter than the sampling interval. If this is not the case, the sampling
rate must be increased with respect to the filter cut-off frequency (see
Section 10.4.2., Sampling at Acquisition Time).
False Events
The probability of detecting false events depends on the amount and
spectrum of noise in the system, the filter characteristics and the rate
of sampling (French et al., 1990).
Multiple Channels
The presence of multiple channels complicates the determination of the
kinetic behavior of a channel. If a record shows a transition from two
open channels to one open, it cannot be determined if the transition
was due to the closing of the first or the second channel. A number of
methods have been proposed to deal with this ambiguity (French et al.,
1990). As a precaution, the amplitude histogram of the raw data can be
inspected to determine if multiple channels are present.
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Data Analysis
Histograms
The simplest histogram is one in which a series of bins are defined,
each of which has an associated upper and lower limit for the quantity
of interest, for example, dwell time. Each dwell time will then fall into
one of the bins, and the count in the appropriate bin is incremented by
one. In the cumulative histogram, a bin contains the number of
observations whose values are less than or equal to the upper limit for
the bin.
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Data Analysis
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Data Analysis
Amplitude Histogram
Amplitude histograms can be used to define the conductances of states
of single channels. Two kinds of amplitude histograms are common:
point histograms and level histograms. The former use all the acquired
data points; they are useful mainly for examining how well-behaved
is a data set. Abnormally wide distributions may result from high noise
if the signal were over-filtered, or if baseline drift had been significant.
Similarly, the number of peaks will indicate the presence of multiple
channels or subconductance states. The all-points histogram will
probably not be useful for determining the conductances unless
baseline drift is small. Level histograms use only the mean baselinecorrected amplitudes associated with each event in the events list.
Such histograms can be fitted to the sum of one or more Gaussian
functions in order to estimate conductances.
Fitting to Histograms
Amplitude and dwell time histograms can be fitted by appropriate
functions, usually sums of several Gaussian functions for the former
and sums of exponentials for the latter (see The Chi-Square Function
(Least-Squares Method) on page 253). The time constants and
amplitudes can be related to the parameters of several single-channel
models, but this will not be described here. Histogram bins containing
zero counts should usually be excluded from a fit because the chisquare function (see below) is not defined when a bin i contains Ni = 0
counts and therefore has i = 0. Alternatively, adjacent bins can be
combined to yield a nonzero content.
Fitting
Reasons for Fitting
Fitting a function to a set of data points, such as a histogram or a time
series, may be done for any of the following reasons:
1. A function could be fitted to a data set in order to describe its
shape or behavior, without ascribing any biophysical meaning
to the function or its parameters. This is done when a smooth
curve is useful to guide the eye through the data or if a function
is required to find the behavior of some data in the presence of
noise.
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Data Analysis
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253
Data Analysis
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Methods of Optimization
Optimization methods are concerned with finding the minimum of a
function (for example, the chi-square) by adjusting the parameters. A
global minimum, for example, the absolute minimum, is clearly
preferred. Since it is difficult to know whether one has the absolute
minimum, most methods settle for a local minimum, for example, the
minimum within a neighborhood of parameter values. A number of
algorithms have been developed to find such a minimum. For example,
to find time constants and coefficients in an exponential fit, pCLAMP
software allows the user to choose between the following:
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Data Analysis
References
Colquhoun, D. and Sigworth, F.J. Fitting and Statistical Analysis of
Single-Channel Records. in Single-Channel Recording. Sakmann, B. and
Neher, E., Eds. Plenum Press, New York, 1983.
Colquhoun, D. and Hawkes, A.G. The Principles of the Stochastic
Interpretation of Ion-Channel Mechanisms. in Single-Channel
Recording. Sakmann, B. and Neher, E., Eds. Plenum Press, New York,
1983.
Dempster, J. Computer Analysis of Electrophysiological Signals.
Academic Press, London, 1993.
Eadie, W.T., Drijard, D., James, F.E., Roos, M, Sadoulet, B. Statistical
Methods in Experimental Physics. North-Holland Publishing Co.,
Amsterdam, 1971.
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Data Analysis
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Noise in Electrophysiological
Measurements
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259
Thermal Noise
Thermal noise results from random motion of thermally excited charge
carriers in a conductor. It is often referred to as Johnson noise or
Nyquist noise. For a resistor, thermal noise can be represented as a
series voltage noise source or a parallel current noise source as
illustrated in Figure 11-1. These two representations are equivalent.
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or
or
Resistance
Value
1 kHz
V noise
(V rms)
10 kHz
V noise
(V rms)
1 kHz
I noise
(pA rms)
100
0.040
0.126
400
1260
1.26
12.6
1 k
0.126
0.40
126
400
4.0
4.0
10 k
0.4
1.26
40
126
12.6
1.26
100 k
1.26
4.0
12.6
40
40
0.4
1 M
4.0
12.6
4.0
12.6
126
0.126
10 M
12.6
40
1.26
4.0
400
0.040
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Resistance
Value
1 kHz
V noise
(V rms)
10 kHz
V noise
(V rms)
1 kHz
I noise
(pA rms)
100 M
40
126
0.40
1.26
1260
0.0126
1 G
126
400
0.126
0.40
4000
0.004
10 G
400
1260 0
0.04
0.126
12600
0.0013
100 G
1260
4000
0.013
0.040
40000
0.0004
Shot Noise
Shot noise arises when current flows across a potential barrier, for
example, a p-n junction in a semiconductor device. Since potential
barriers are not present in simple resistive elements, resistors do not
display shot noise. Over a bandwidth B, the rms value of the shot noise
current is given by:
1.
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1 A
18
57
100 nA
5.7
18
10 nA
1.8
5.7
1 nA
0.57
1.8
100 pA
0.18
0.57
10 pA
0.057
0.18
1 pA
0.018
0.057
0.1 pA
0.0057
0.018
Dielectric Noise
An ideal lossless capacitor does not produce thermal noise. However, all
real dielectric materials display some loss that results in the generation
of thermal noise. For dielectrics with relatively low losses, the spectral
density of this noise SD2 can be described in terms of the dissipation
factor D and the capacitance CD of the dielectric:
For the best solid dielectrics (for example, quartz, sapphire and some
ceramics), D is on the order of 10-5 to 10-4. For poorer (lossier)
dielectrics D can be much higher; for example, some thermosetting
plastics have D in the range of 0.01 to 0.1. For some glasses used to
fabricate patch pipettes, D is at least 0.01. The dissipation factor has
some frequency dependence, although in the important range from
about 1 kHz to 100 kHz it is usually reasonable to approximate it as a
constant. D also shows some temperature dependence (typically
decreasing with lower temperatures), although again in the usual range
of temperatures it may be considered constant.
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Excess Noise
Excess noise can broadly be defined as any noise that is present in a
circuit element in addition to the fundamental noise mechanisms
already described. For example, in the presence of direct current all
resistors exhibit low-frequency noise whose power spectral density
varies inversely with frequency. This noise is present in addition to the
thermal noise of the resistor and is usually referred to as 1/f noise.
Different resistor types display different amounts of 1/f noise, with
metal film and wirewound types showing the least excess
low-frequency noise and carbon composition resistors showing the
most. The PSD (in V2/Hz) of this excess noise rises with decreasing
frequency essentially as 1/f. Its magnitude is directly proportional to
the DC current flowing through the resistor. Semiconductor devices also
exhibit 1/f noise.
High-value (gigaohm range) resistors, such as those used as the
feedback element in resistive-feedback patch-clamp headstages, also
display a form of excess noise that rises with increasing frequency.
Generally, such resistors only achieve their thermal noise level (which is
the minimum possible) in the frequency range from about 10 Hz to
1 kHz. At low frequencies, 1/f noise is observed, and at high
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Amplifier Noise
The intrinsic noise of an operational amplifier can be described by an
equivalent input voltage noise En in series with the negative (-) input
and an equivalent input current noise In between the positive (+) and
negative (-) inputs (see Figure 11-2). These noise sources have been
referred to the input for convenience of analysis. It should be noted,
however, that they are measured at the output. For example, in an
open-loop situation (such as shown in (Figure 11-2), if the inputs are
grounded, the voltage at the negative (-) input will be zero (ground). En
must be inferred by measuring the output noise voltage and dividing it
by the open-loop gain (both are frequency dependent) of the amplifier.
On the other hand, in a closed-loop situation (which is always the case
when using operational amplifiers, as illustrated in Figure 11-3), En will
actually appear at the negative (-) input due to the action of the
feedback loop.
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267
Electrode Noise
Noise associated with recording electrodes is significant and often
dominant in most electrophysiological measurements. In
microelectrode voltage clamps and the whole-cell variant of the patch
voltage-clamp technique, the thermal voltage noise of the electrode is
of greatest importance since this noise will exist across the cell
membrane and produce large levels of current noise in conjunction with
the membrane capacitance. In patch single-channel measurements
using the patch clamp, the voltage noise of the electrode is also
important; but here the situation is further complicated by such factors
as the dielectric noise of the pipette that produces a major source to
overall noise. We will consider noise associated with the electrode in
single-channel recording and whole-cell patch recording separately,
beginning with the single-channel recording.
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Dielectric Noise
The basic characteristics of dielectric noise have already been described
above (see equations (9) and (10)). Dielectric noise of the pipette can
be a major contributor to total noise in patch voltage clamping; in some
situations it can be the dominant noise source. The dielectric noise
arising from the pipette depends on the dissipation factor D of the glass
used to fabricate the pipette, on the pipette capacitance (CD in
equations (9) and (10)), and on the presence of Sylgard coating. The
dissipation factor D of glasses other than quartz that have been
successfully used for patch pipettes generally falls in the ranges of
0.0010.01. The dissipation factor for quartz is variously reported to be
10-5 to as much as 4 x 10-4. For Amersil T-08 quartz, which has been
used in all of the preliminary tests of quartz pipettes described below,
the reported dissipation factor is 10-4. For uncoated pipettes the value
of CD is determined by the dielectric constant of the glass, the ratio
of the outer diameter (OD) to the inner diameter (ID) of the pipette,
the depth of immersion of the pipette into the bath, and to some extent
by the pulling characteristics of the glass and the geometry near its tip.
The dielectric constant ranges from as little as 3.8 for quartz to more
than 9 for some high-lead glasses. Typical glass capillaries used in the
fabrication of patch pipettes have an OD of 1.65 mm and an ID of
1.15 mm; thus OD/ID 1.43. If it is assumed that these proportions
(OD/ID = 1.43) are maintained as the glass is drawn out in pulling,
then for uncoated pipettes the value of CD will be approximately
0.15 pF per mm of immersion into the bath. For thick-walled glass
with OD/ID = 2, this value would fall to 0.08 pF per mm of immersion;
while for thin-walled glass with OD/ID = 1.2 the capacitance would
increase to about 0.30 pF per mm.
Actual measurements with uncoated or lightly Sylgard-coated pipettes
fabricated from glasses with known and immersed to a depth of
23 mm indicate that these values often underestimate pipette
capacitance; therefore, the dielectric noise is also underestimated. This
is probably due to non-uniform thinning near the tip and to some
uncertainty as to the true depth of immersion (due to a meniscus of
solution around the pipette). For example, using the popular Corning
7052 glass, which has = 4.9 and with OD/ID 1.43, it is not
uncommon to measure a pipette capacitance as high as 3 pF, for
example, about twice the predicted value, when an uncoated pipette or
a pipette very lightly coated with Sylgard is immersed at a depth of
2 mm.
Despite this precautionary note, it is clear that, all else being equal, the
value of CD varies linearly with the dielectric constant of the glass.
Equation (10) indicates that if the depth of immersion and the OD/ID
ratio are constant, the rms noise for a given bandwidth arising from the
lossy dielectric characteristics of pipettes fabricated from various
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the formation of a thin film of solution that will creep up the outer wall
of an uncoated pipette. Such a film can produce very large amounts of
noise in uncoated pipettes. Sylgard coating not only virtually eliminates
this noise source but also thickens the wall of the pipette thereby
reducing its capacitance. The dielectric constant of Sylgard #184 is 2.9
and its dissipation factor is 0.002, which is lower than that of most
glasses that have been used for patch pipette fabrication. Thus, coating
with Sylgard will reduce dielectric noise of patch pipettes. It is expected
that the improvement in noise associated with Sylgard coating will be
greatest for glasses with a high D product (for example, soda lime
glass); this has been confirmed experimentally. Improvement of noise
should be less for glasses with very low values of D, but coating with
Sylgard will reduce the dielectric noise of all glasses. The effects of
Sylgard coating on noise are, however, difficult to quantify theoretically
primarily because the thickness of the coating is usually not uniform. In
particular, it is difficult to achieve a very thick coating very near the tip.
Experimental tests of the noise properties of patch pipettes fabricated
from 19 different kinds of glass (see Chapter 11: Noise in
Electrophysiological Measurements on page 259) have confirmed the
general conclusions described above. With few exceptions, the noise
attributable to the pipette is inversely correlated with the D product of
the glass. In addition, thicker-walled pipettes and shallow depths of
immersion reduce noise for any particular glass type. Sylgard coating
has its greatest effect on the glasses with the poorest inherent
electrical properties, but it is important to remember that such coating
is necessary for all types of glass.
It has been obvious for some time that pipettes fabricated from quartz
should produce only very small amounts of dielectric noise due to the
low dielectric constant of quartz ( = 3.8) and, more importantly, its
extremely low dissipation factor (D 10-5 10-4). However, due to the
high melting point of quartz ( 1600 C), only with the advent of
automated laser pullers has it become practical to pull patch pipettes
from quartz tubing. A quartz pipette with D = 10-4 that is immersed to a
depth of 2 mm (again assuming 0.2 pF per mm of immersion) would
be predicted to produce only about 0.03 pA rms of dielectric noise in a
bandwidth of 10 kHz (-3 dB, 8-pole Bessel filter); for D = 10-5 this
value would fall to 0.01 pA rms. Preliminary measurements using
Amersil T-08 quartz suggest that the amount of dielectric noise in this
situation is closer to 0.040.05 pA rms. A more detailed discussion of
preliminary estimates of the noise properties of quartz pipettes is
provided below.
Dielectric noise is probably the largest source of noise for pipettes
fabricated from all glasses other than quartz. For pipettes fabricated
from quartz, due to its very low dissipation factor, sources of noise
other than dielectric noise are expected to dominate total pipette noise
(See Noise Properties of Quartz Patch Pipettes on page 275).
To summarize, dielectric noise can be minimized by using thick-walled
glasses with low values of D and coating the pipette with Sylgard #184.
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The effects of Sylgard coating are greatest for glasses with relatively
poor electrical properties. For excised patches, dielectric noise can be
minimized by withdrawing the tip of the pipette as close as possible to
the surface of the bath.
It should be noted that dielectric noise will also contribute to the noise
associated with the holder. For an Axopatch 200A amplifier with an
open circuit noise of 0.06 pA rms in a 5 kHz bandwidth, total noise
should not increase to more than about 0.07 pA rms in this bandwidth
when the Axon-supplied polycarbonate holder is attached. Part of this
noise increment is due to the fact that the holder adds about 11.5 pF
of capacitance to the headstage input. The rest of the increment in
noise associated with the holder is presumably dielectric noise, which
can be estimated to account for roughly 0.03 pA rms in a 5 kHz
bandwidth.
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273
274
Although the dielectric characteristics of the specific Silicone fluid used in these
experiments is not available, Table 2.5 of Electronics Designers' Handbook
(1977, L. Giacoleto ed., McGraw-Hill) lists a dissipation factor (D) of 8.5x10-5 at
1 kHz and 2x10-5 at 100 kHz for Silicone fluid (methyl- or ethyl-siloxane
polymer); the dielectric constant is 2.68.
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where e2e = 4kTRe is the thermal voltage noise of the pipette. The rms
noise arising from this mechanism from DC to a bandwidth B is then
3.
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Note, however, that the PSD for quartz pipettes is lower than that of pipettes
made from other glasses at all frequencies measured. At sufficiently high
frequencies distributed R-C noise should dominate the total noise for pipettes
fabricated from any type of glass. However, for quartz pipettes the PSD of
distributed R-C noise should exceed that of dielectric noise by 1 kHz; for pipettes
made from 7760, distributed R-C noise should not exceed dielectric noise up to
1020 kHz, and for soda lime (for example, 0080) pipettes this frequency might
be closer to 100 kHz or more. Distributed R-C noise depends on the pipette
geometry and a variety of other factors already described; the only influence of
the glass type itselfaccept in so far as it effects the geometry which can be
pulledis its dielectric constant.
277
Seal Noise
The membrane-to-glass seal that is essential to the patch voltageclamp technique is the final source of noise associated with the pipette
that will be considered here. Seal noise is perhaps the least understood
source of noise in the patch clamp technique. The power spectral,
S2sh(f), of the noise arising from the seal for zero applied voltage is
given by:
where Re{Ysh} is the real part of the seal admittance Ysh. The minimum
estimate of seal noise results from the assumption that
Re{Ysh} = 1/Rsh, where Rsh is the DC seal resistance. If this assumption
is correct, then for a 10 kHz bandwidth, seal resistances of 1, 10 and
100 G would produce noise of 0.4, 0.13 and 0.04 pA rms,
respectively. Since values of Rsh in the range of 100200 G are not
uncommon, this would imply that the noise associated with a very tight
seal would be small in comparison with other sources of patch-clamp
noise. However, it is possible that the PSD of seal noise may rise above
the minimum level given by 4kT/Rsh as frequency increases (for
example, the real part of the seal admittance may increase with
frequency) due to the capacitance of the glass and the membrane
which make up the wall of the seal. Unfortunately, we have no good
theoretical basis upon which to estimate Ysh since the precise nature of
the membrane-glass seal is not known.
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where e2s = 4kTRs and sr = RsrCm and Rsr is the residual (i.e.,
uncompensated) series resistance, for example, if Rs = 10 M then for
series-resistance compensation levels of 50%, 70% and 90%, Rsr will
be 5 M, 3 M and 1 M, respectively. It should be noted that for
100% series-resistance compensation, equation (16) reduces to
. This emphasizes that series-resistance compensation (if it is
properly designed) only restores the noise to the level that would have
resulted from the thermal voltage noise of Rs in series with Cm in the
absence of the filtering effect of Rs mentioned above. The rms noise
arising from Rs and Cm over a bandwidth from DC to B Hz can be
obtained by integrating equation (16) over f from 0 to B.
Patch clamps commonly use (switch in) a 500 M feedback resistor for
whole-cell voltage clamping. The noise of this resistor dominates opencircuit headstage noise up to bandwidths of about 10 kHz. However, for
typical cells the noise arising from Rs and Cm will be larger than that of
the headstage for all bandwidths above a few hundred Hz. Even for
relatively ideal situations in terms of noise (for example, a small cell
with Cm = 5 pF voltage clamped through Rs = 5 M), the noise of a
500 M feedback resistor will not dominate total noise for bandwidths
above about 1 kHz. Figure 11-4 shows the noise PSD and rms noise for
a typical whole-cell voltage clamp situation with Rs = 10 M and
Cm = 33 pF (these are the values used in the Model Cell provided with
the Axopatch-1 and the Axopatch 200 series of patch clamp amplifiers).
The top panel shows the noise PSD (as computed from equation 16 plus
headstage noise) for series-resistance compensation levels of 0%,
50%, 70% and 90%; the noise PSD of an open-circuit headstage alone
is shown for comparison. It is instructive to derive an expression for the
high frequency plateau of these PSDs (from equation 16). If the
fraction of series-resistance compensation is defined as and
= 1 (for example, for 80% compensation = 0.2; also note
that Rsr = Rs), then as f , S2wc(f) 4kT/(2R). Even without
series-resistance compensation ( = 1) the high frequency plateau is
4kT/Rs; for example, 50 times larger than that of a 500 M resistor.
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The lower panel of Figure 11-4 shows the total rms noise as a function
of bandwidth for the same cell parameters with Rs compensation levels
of 0%, 50%, 70% and 90%; the rms noise of the open circuit
headstage is also shown. A bandwidth of only about 200 Hz the total
noise is twice that of the headstage alone; therefore, even if a
headstage with negligible noise had been used, at this bandwidth total
noise would only decline to about 70% of the value shown. As the
bandwidth of current measurement increases, the noise of the
headstage itself becomes progressively more negligible; with 90%
compensation at a bandwidth of 1 kHz, a headstage with no noise
would have reduced total noise by less than 1% (recall that the noise of
the headstage and the Rs-Cm noise considered here are not correlated
and, therefore, add in an rms fashion). It should also be pointed out
that the bandwidths in this figure refer to the setting of an external
filter (a perfect brick-wall filter has been assumed, see Filtering on
page 287). But it is important to realize that the actual bandwidth of
current measurement is limited to 1/2RsrCm (1 pole RC filter). Without
series-resistance compensation, in this example the bandwidth is only
about 480 Hz and the use of external filter bandwidths much above this
will only add noise, not new signal information. The effective bandwidth
increases with increasing series-resistance compensation, reaching
nearly 5 kHz with 90% compensation.
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Digitization Noise
Noise arising from digitization is often ignored. Sometimes this is
justified since such noise can be negligible with respect to other
sources of noise. However, in some situations this potential source of
noise can become significant. In order to ensure that this noise remains
negligible one needs to understand the types of noise that can arise
from digitization and to use analog-to-digital converters, preamplifiers,
filters, etc. that are appropriate for the measurement of the signal.
Quantization is the approximation of each value of a signal by an
integer multiple of an elementary quantity , which is the quantizing
step. For a 12-bit analog-to-digital converter (ADC) with full-scale
range (FSR) of 10 V, = 20 V/212 = 4.88 mV; for a 16-bit converter
with the same FSR, = 305 V. This approximation leads to the
addition of a noise signal, called quantizing noise, to the original signal.
When the signal being digitized is reasonably large relative to the
quantizing step , the power of the quantizing noise can usually be
approximated by:
For a 12-bit ADC with a 20 V FSR this noise value is 1.41 mV rms or
about 8.5 mV peak-to-peak.
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Aliasing
A signal can be determined completely by a set of regularly spaced
samples at intervals T = 1/fs only if it does not contain components with
a frequency equal to or greater than fs/2. This is basically a statement
of the sampling theorem; fs is just the sampling frequency mentioned
above. The term fs/2 will be denoted by fn and is often called the
Nyquist frequency or folding frequency for reasons that will be
described below. Another way of putting this is that for sampled data,
frequency is only defined over the range from 0 to fn; components with
frequencies higher than fn cannot be described (at least 2 points per
cycle are needed to uniquely define a sine wave). Obviously there is
nothing (other than good sense) that will stop one from digitizing a
signal with frequency components extending many times beyond fn.
However, digitizing frequency components of the signal that lie above fn
will result in folding back of these higher frequency components into
the frequency range from 0 to fn, consequently producing aliases.
The term fn is referred to as the folding frequency because the
frequency axis of the power spectral density will fold around fn in a
manner similar to folding a road map or a carpenters scale. This folding
effect is illustrated in Figure 11-5; frequency components above fn are
shifted to lower frequencies (in the range 0 to fn). If fx is the frequency
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Filtering
The above discussion naturally leads to a brief discussion of filtering. In
general, the bandwidth of a filter is selected to reduce noise to
acceptable levels so that the desired signal can be adequately
observed. As described above, filtering prior to digitization is also
necessary to prevent aliasing. If the signal to be measured is large in
comparison with the background noise, then the filter bandwidthand
the appropriate digitization ratecan be chosen on the basis of the
desired time resolution; wider bandwidths allow more rapid events to
be resolved. However, in many electrophysiological measurements very
wide bandwidths will result in background noise levels that will obscure
the signals of interest. In such situations it is necessary to make
compromises between the amount of noise that can be tolerated vs.
the time resolution that is achieved after filtering.
There is an interestingalthough perhaps unfortunaterelationship
between a function and its Fourier transform: as the function gets
narrower its transform becomes wider. The impulse response of a filter
(time domain; note that the integral of the impulse response is the step
response) and its transfer function (frequency domain) are a Fourier
transform pair. There are limits on the degree to which signals can be
simultaneously concentrated in both the time and the frequency
domain (in fact, if stated formally, this is the uncertainty principle in the
units we are using). In practical terms this means that a filter with a
narrow impulse response, and therefore a rapid rise time, will have a
rather gradual roll-off in the frequency domain. Conversely, a filter with
a sharp cut-off in the frequency domain will have a more spread-out
impulse response and slower rise time.
Among commonly used filters, those that provide the best resolution
with little or no overshoot and ringing of the step response are the
Gaussian and Bessel filters (as the order of a Bessel filter increases it
more closely approximates a Gaussian filter). A true Gaussian filter is
easy to implement digitally, but is not often produced as an analog filter
(although passive filters that are a good approximation of this type can
be constructed). Bessel filters are more commonly used in analog
applications. The basic characteristics of both filter types are quite
similar. A Gaussian filter has an impulse response with the shape of the
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where f1, f2, f3 ... are the -3 dB bandwidths of the individual filters in
series. Of course, the same expression holds for a series combination of
analog filters. The disadvantage to this general approach is that initial
data storage will be increased due to the high digitization rate; once the
data has been digitally filtered it can also be decimated if desired.
Holder
The pipette holder contributes noise by increasing the capacitance at
the headstage input; some dielectric noise must also be expected. The
unshielded polycarbonate holders provided by Molecular Devices by
themselves will only increase the headstage noise by about 10% above
its open circuit value even for the lowest noise capacitive-feedback
devices. For example, for the Axopatch 200B amplifier with an
open-circuit noise of 0.060 pA rms in a 5 kHz bandwidth (as shown on
the panel meter), the total noise with the holder attached should not
increase beyond 0.070 pA rms (note that larger noise increments
probably mean that the holder needs to be cleaned), and will often be
as low as 0.065 pA rms. Inserting a saline-filled electrode into the
holder will further increase noise even while the electrode tip is in air.
With a saline-filled electrode, the noise of the Axopatch 200B with an
open-circuit noise of 0.060 pA in a 5 kHz bandwidth will generally
increase to about 0.075 pA rms.
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Electrode
The noise associated with the patch pipette is determined by the type
of glass used, the wall thickness of the glass, the pipette geometry, the
use of Sylgard coating, and the depth of immersion into the bath.
Dielectric noise is probably the dominant source of noise associated
with the electrode for all glasses except quartz. For glasses with lower
D productsmost notably quartzit can be expected to become a
much smaller component of overall noise. Additional noise will arise
from the distributed resistance and capacitance of the pipette. For the
best glasses presently in common use, this source of noise is probably
somewhat less than the dielectric noise of the glass; for very low-loss
glasses, such as quartz, it could be the dominant source of pipette
noise. A small amount of noise will also result from the thermal noise of
the pipette resistance in series with the patch capacitance; only under
certain situations (a large patch area) will this Re-Cp noise become
significant.
Theoretical and experimental results indicate that a pipette fabricated
from the lowest-noise glasses (other than quartz) used to date
(Corning #7760, #8161) with a moderate coating of Sylgard will
produce noise of about 0.2 pA rms in a 10 kHz bandwidth (8-pole
Bessel filter) for an immersion depth of about 2 mm. Under favorable
circumstances, this value can be cut at least in half when the tip of the
pipette is withdrawn to within 100200 m of the surface of the bath.
For pipettes fabricated from quartz, preliminary results indicate that for
a 1 mm depth of immersion, noise of somewhat less than 0.1 pA rms
can be expected in a bandwidth of 10 kHz; with the very small
immersion depth possible (10 m or less) with the Silicone-fluid
technique described above, it can be estimated that the noise
contributed by a quartz pipette falls to less than half of this value.
Results obtained for quartz have involved pipettes with relatively long
narrow shanks and resistances of roughly 10 M. Such a geometry is
not ideal for achieving the lowest possible noise. As techniques for
pulling quartz pipettes improve, and as other grades and suppliers of
quartz are investigated, further improvements are likely.
Seal
The noise associated with the seal is less easily predicted. Certainly a
minimum estimate of this noise in a bandwidth B is given by
(4kTB/Rsh)1/2, for example, the thermal current noise of the DC seal
resistance, Rsh. For excellent seals in the range of 50200 G this
would mean that the minimum noise attributable to the seal is in the
range of 0.030.06 pA rms in a 10 kHz bandwidth. Recent data
suggests that under favorable circumstances seal noise may be as low
as these predicted values. However, experience indicates that there is a
good deal of variability in the noise of patches even when the seal
resistances are very high and all other precautions necessary to
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Further Reading
Rae, J.L., Levis, R.A., A method for exceptionally low noise singlechannel recording. Pflugers Arch. 420, 618620, 1992.
Rae, J.L., Levis, R.A., Quartz pipettes for single-channel recording.
Biophys. 64, A201 (P394), 1993.
Sakmann, B. and Neher, E., Eds. Single-Channel Recording. New York:
Plenum Press, 1983.
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General
Following are terms commonly used in electrophysiological
measurements.
Thermal Noise
All resistors generate thermal noise. Thermal noise can be represented
by a mean square voltage generator (e2n) in series with a noiseless
resistor:
2-Pole
4-Pole
Butterworth Butterworth
DC-100 Hz
5.07
4.27
4.11
4.06
DC-1 kHz
16.0
13.5
13.0
12.8
DC-10 kHz
50.7
42.8
41.1
40.6
DC-100 kHz
160
135
130
128
Bandwidth
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Infinitely
Sharp
295
Filters
Low-pass filters are commonly used in electrophysiology to reduce
noise. The important parameters of a low-pass filter are the -3 dB
frequency, the type and the order.
The -3 dB frequency (f-3) is the frequency where the voltage response
falls to 2 (0.707). Some manufacturers specify the cutoff or
bandwidth based on the phase response or an asymptotic
approximation to the high and low frequency amplitude response.
Therefore when using a new filter you should check that the specified
settings refer to the -3 dB frequency.
The three types of low-pass filters commonly used in electrophysiology
are the Bessel, Butterworth, and multiple coincident pole (RC) types.
The most important differences are:
1. When driven by a step voltage, the RC filter has no overshoot,
the Bessel filter has < 1% overshoot and the Butterworth filter
has around 10% overshoot.
2. When driven by a noisy source, RC filters show the least
rejection of the noise at frequencies above f-3, Bessel filters
show moderate rejection and Butterworth filters show the most.
The order of a filter refers to the number of poles. Each
resistor-capacitor section contributes one pole (inductors are rarely
used for bandwidths < 100 kHz). Thus 4th-order, 4-pole, and 4 RC
sections all refer to the same thing.
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Microelectrode Amplifiers
Noise magnitude is very sensitive to the measurement technique.
Specifications should state:
1. -3 dB bandwidth and order of the filter used in the
measurement circuit. Typically the noise will look 2030% better
if a fourth-order low-pass filter is used instead of a first-order
low-pass filter.
2. Microelectrode bandwidth. The capacitance neutralization should
be adjusted so that the -3 dB bandwidth of the microelectrode is
the same as the -3 dB bandwidth of the measurement circuit.
Voltage-Clamp Noise
Almost impossible to specify. Depends on cell mode, electrode model,
capacitance neutralization setting, electrode interactions, electrode
resistances, current measurement techniques, bandwidth of
measurement circuit and clamp gain.
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Input Capacitance
In a circuit using capacitance neutralization and other compensation
techniques, it is meaningless to specify a value for the input
capacitance. The efficacy of the neutralization circuit depends on the
magnitude of the electrode resistance and the measurement technique.
For an electrode with resistance R and an exponential response to a
step input, the effective input capacitance can be estimated from:
Patch-Clamp Amplifiers
Bandwidth
The bandwidth of a patch-clamp headstage is usually measured by
injecting current into the headstage input through a small capacitor. No
holder or electrode is attached to the input.
The bandwidth during an experiment is usually much lower. It is
typically limited by the electrode resistance and membrane
capacitance, as well as by stray capacitances. For example, if a 5 M
electrode is used to clamp a whole cell having 20 pF of membrane
capacitance, the time constant for measuring membrane currents is
100 s (5 M x 20 pF). This corresponds to a -3 dB bandwidth of
1.6 kHz, far below the 100 kHz that may be specified. 80%
series-resistance compensation would improve this to 8 kHz.
Nevertheless, it is still better to have a headstage with very wide
bandwidth because in some cases series-resistance compensation may
work better.
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Noise
The ideal RMS current noise in selected bandwidths is shown in
Table A-2 for various feedback resistors. These figures assume ideal
resistors (which only have thermal noise) and noiseless electronics.
Table A-2 rms Current in Picoamps.
BANDWIDTH 500 M
1 G
5 G
10 G
50 G
DC-1 kHz
0.182
0.128
0.057
0.041
0.018
DC-3 kHz
0.331
0.234
0.099
0.074
0.033
DC-10 kHz
0.574
0.406
0.181
0.128
0.057
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